Note: Descriptions are shown in the official language in which they were submitted.
~ 17~6~
X-5155 -1-
BIS-TETRAZOYL~THYL SUBSTITUTED
CEPHALOSPORI~ ANTIBIOTTCS
7~-Acylamino cephalosporin compounAs sub-
stituted in the 3-position with a 1-(lH-tetrazol-5-
yl-methyl)-lH-tetrazol-5-ylthiomethyl group of the
formula
-CH2-S-
~H2 -
., 4
or a 2-(lH-tetrazol-5-yl-methyl)-2H-tetrazol-5-yl-
thiomethyl group of the ormula
_c~2-S- ~ /N-CH2 \N/
,~ 1
: I K4
~¦ 20 where in each of the above formulas R~ is hydrogen, or
Cl-C3 alkyl, are broad spectrum antibiotics which
inhibit the growth of microorganisms pathogenic to man
and animals. The compounds are useful when adminis-
. tered parenterally in suitable pharmaceutical dosage
:- 25 forms. Compounds of the invention wherein the 3 substitu-
.j ~nt is the l-(lH-tetrazol-5-yl-methyl)-lII-tetrszol-5-yl-
'~ thiomethyl group provide effective blood levels of
extended duration in animals.
~, The bis-tetrazolmethyl substituted cephalo-
sporlns of this invention are prepared by severai
'~
: - - . .. , ~ ,
,, :
~- ..
, ~ , : I ,:-: - .
.
., . ~ . .
~:;.,.~ . ,.
-i
,
.
:
~ ~ ~ 4 6 6 ~
X-51~5 -2-
preparative methods. For example, 7-aminocephalospo-
ranic acid (7-ACA) is reacted with the appropriate
bis-tetrazolmethyl thiol to form the 7-amino-3-bis-
tetrazolmethyl thiomethyl-3-cephem-4-carboxylic acid,
and the latter intermediate is acylated at the 7-posi-
tion amino group to provide a 7-acylamino substituted
cephalosporin. Alternatively, a 7-acylaminocephalo-
sporanic acid can be reacted with the appropriate
bis-tetrazolmethyl thiol to provide a compound of the
invention. Lastly, a 7-acylaminocephalosporanic acid
is reacted with l-cyanomethyl-lH-tetrazol-5-yl-thiol
to provide the corresponding 7-acylamino-3-(1-cyano-
methyl-lH-tetrazol-5-yl-thiomethyl)-3-cephem-4-carboxyllc
acid intermediate, and the latter is reacted at the
cyano group of the 3-tetrazole moiety with an appropri-
ate azide to form the bis-tetrazolmethyl compound o~
~ the invention.
¦ The invention also provides the bis-tetrazol-
methyl thiomethyl substituted cephalosporin nucleus
having an unacylated amino group in the 7-position.
This 7-amino nucleus compound is useful in preparing
the 7-acylaminocephalosporins of the invention as
described above.
Cephalosporin antibiotics having various
heterocyclic thiomethyl substituents in the 3-position
are known. For example, Takano et al., U.S. Patent No.
¦ 3, 516, 997, describe tetrazolthiol and thiadiazolthiol
, substituted cephalosporins having a heterocyclic acyl-
amino group in the 7-position; Ryan, U.S. Patent ~o.
3,641,021 describes tetrazolthiol and thiadiazolthiol
~ ''"'' ' . :
, .
~ 17~668
I X-5155 _3_
substituted cephalosporins having a mandeloyl or phenyl-
glycyl substituent in the 7-position; Breuer, et al.,
U.S. Patent No. 4,110,535, describe certain cephalospo-
rin compounds having a 3-oxopyridazinylthiomethyl
substituent in the 3-position; Berges, U.S. Patent No.
4,107,173, describes cephalosporin compounds having as
the 3' substituent a 3-sulfomethyl-1,2,4-triazol-5-
ylthiomethyl substituent; Toshiyasu, et al., U.S.
Patent No. 4,103,008, describe cephalosporin compounds
substituted in the 7-position by a dioxopiperazine-l-
yl-carbonylamino substituent and in the 3-position by a
substituted or unsubstituted 1,3,4-thiadiazol-2-
ylthiomethyl substituent; Berges, U.S. Patent No.
4,101,656, describes 7-acylamino substituted cephalo-
1 15 sporins having ~ tetrazolthiomethyl substituent in the
i 3-position which is substituted with an alkylsulfonamido-
alkyl group; Gottstein, et al., U.S. Patent No. 4,100,346,
describe certain o-aminophenylacetamido substituted
~j cephalosporins having a tetrazolthiomethyl substituent
~ 20 in the 3-position which is substituted with a carboxy-
i ethyl or carboxypropyl substituent; Numata, et al.,
U.S. Patent No. 4,080,498, disclose a 2-aminothiazol-
i acetamido cephalosporin substituted in the 3-position
with a heterocyclic thiomethyl group; Dunn, et al.,
U.S. Patent No. 3,968,226, describe numerous 3-hetero-
cyclicthiomethyl substituted cephalosporins; Naito, et
al., U.S. Patent ~o. 4,082,912, disclose 7-acylamido-
cephalosporins having the fused heterocyclic, (2-
carboxyalkyl)-2,3-dihydro-S-triazolo~4,3-b]pyridazin-
3-one-6-ylmethyl group in the 3-position; British
:
... .. . . ... . ..
" ' . ~ .
,,
J
1 :~74~
X-5155 _4_
Patent Speci~ication No. 1,525,626 describes certain
cephalosporin compounds having a l-carboxymethyl-lH-
tetrazol-5-thiomethyl substituent in the 3-position;
Dunn, et al., U.S. Patent No. 3,985,739, describe
7-phenylglycyl or 7-mandeloyl substituted cephalosporins
having in the 3-position a 1,3,4- or a 1,2,3-triazol-
thiomethyl substituent; Naito, et al., U.S. Patent No.
3,985,738, describe 7-mandelamidocephalosporins having
in the 3-position a tetrazolo[4,5-b]pyridazin-6-ylthio-
methyl substituent; and Barth, U.S. Patent No. 3,966,719,
discloses cephalosporin compounds wherein the 4-
carboxy group is replaced with a tetrazol-5-yl group.
3-Heterocyclicthiol substituted methyl cepha-
losporins are generally prepared by the displacement of
the acetoxy group of a 3-acetoxymethyl-3-cephem, or the
halogen of a 3-halomethyl-3-cephem compound, with the
desired heterocyclic thiol. Hatfield, U.S. Patent No.
4,144,391, describes a non-aqueous displacement method
for the preparation of 3-heterocyclicthiomethyl substi-
1 tuted cephalosporins.
j This invention is concerned with broad spec-
trum cephalosporin antibiotics which differ structurally
from the ]snown cephalosporins. In particular, this
invention relates to 7-amino and 7-acylamino cephalo-
sporin compounds substituted in the 3-position of the
dihydrothiazine ring with a bis-tetrazolylmethyl group.
In particular, the bis-tetrazolylmethyl substituents
are the l-(lH-tetrazol-S-ylmethyl)-lH-tetrazol-5-ylthio
group and the isomeric 2-(lH-tetrazol-5-ylmethyl)-2H-
tetrazol-5-ylthio group.
11 17~669
X-5155 -5-
Specifically, the cephalosporin compounds of
this invention are represented by the following general
formula
~ R2
~ t
o~
OOR1 I
lo
wherein R3 is ~ ~ ~
\~4
and R4 is hydrogen, or C1-C3 alXyl;
wherein R is hydrogen or an acyl group derived from a
carboxylic acid;
R} is hydrogen or a carboxy-protecting group which is
readily removed by hydrolytic or hydrogenolysis methods;
~ and
;i R2 is hydrogen or methoxy.
! The term "acyl group derived from a carboxylic
acid" refers to the 6- and 7-acyl portions of the side
chain acylamino substituents of the known penicillin
I and cephalosporin antibiotics. Examples of carboxylic
. acids providing such acyl groups are the alkylcarboxylic
acids such as formic acid, acetic acid, propionic acid,
; n-butyric acid, iso-butyric acid, n-valeric acid,
trimethylacetic acid, caproic acid, n-heptylic acid,
i, .
1 ~7~6~
X-515S -6-
caprylic acid, and the like; the alkenoic acids, for
example, acrylic acid, crotonic acid, and the like;
the substituted alkyl carboxylic acids wherein the
substituent is hydroxy, halogen, or cyano, for example,
chloroacet c acid, bromoacetic acid, bromobutyric acid,
chloropropionic acid, hydroxyacetic acid, gamma-
hydroxybutyric acid, cyanoacetic acid, cyanopropionic
acid, and cyanobutyric acid; the aromatic carboxylic
, acids, such as benzoic acid and substituted benzoic
J 10 acids, for example, the toluic acids, p-chlorobenzoic
acid, 3,4-dichlorobenzoic acid, 3-fluorobenzoic acid,
p-hydroxybenzoic acid, the carboxy-substituted benzoic
acids, for example, phthalic acid and terephthalic acid,
p-aminobenzoic acid, m-aminobenzoic acid, and the
lS acetylated derivatives thereof, 3,4-dihydroxybenzoic
acid, 3-chloro-4-hydroxybenzoic acid, o-methoxybenzoic
acid, p-methoxybenzoic acid, p-ethoxybenzoic ~cid, and
like substituted benzoic acids; the aryl alkanoic acids
such as phenylacetic acid and the substituted phenyl-
20 acetic acids for example, 4-hydroxyphenylacetic acid,
-~ 4-chlorophenylacetic acid, 2-aminomethylphenylacetic
acid, 3-carboxymethylphenylacetic acid and the like;
the aryl alkanoic acids wherein the aryl portion is
separated from the alkyl portion by a hetero atom such
25 as phenoxyacetic acid, and the substituted phenoxyacetic
acids, for example, p-chlorophenoxyacetic acid, p-hydroxy-
i phenoxyacetic acid, phenylmercaptoacetic acid, and the
J. halogenated darivatives thereo~, for example, 3,4-
dichlorophenylmercaptoacetic acid, p-chlorophenyl-
30 mercaptoacetic acid, p-fluorophenylmercaptoacetic acid,
,, . ~ .. ,. ~ .~ .
3 174~
X-5155 -7-
! 3,4-dimethylphenylmercaptoacetic acid, and the like;
the aryl acetic acids, wherein the alkyl portion is
¦ substituted, for example, phenylglycine, mandelic acid,
phenylmalonic acid, -sulfophenylacetic acid, and such
acids wherein the aromatic ring bears a substituent,
for example, halogen, hydroxy, carboxy, alkyl, for
example, methyl trifluoromethyl, alkoxy, for example,
methoxy and ethoxy, amino and substituted amino, such
as acetamido, and like substituted acids; the hetero-
aryl acetic acids, wherein the heteroatom contains oneor more oxygen, nitrogen, or sulfur atoms along with
carbon in the hetero ring, for example, the 5- and 6-
membered heterocyclic aryl acetic acids, such as thio-
pheneacetic acid, furaneacetic acid, pyridylacetic
acid, and the hydroxy-substituted pyridylacetic acids,
I for example, 4-hydroxypyridyl-3-acetic acid, imidazol-
¦ acetic acid, thiazoleacetic acid, oxazoleacetic acid,
¦ 1,3,4-thiadiazolacetic acid, 1,3,4-oxa~iazolacetic
¦ acid, isooxazoleacetic.acid, and the like; and the
benzheterocyclic acetic acids, for example, 2-benzo-
thienyl acetic acid, 2-benzofuran acetic acid, and like
. heterocyclic aryl acetic acids. Acyl moieties repre-
sented by the term "R" in the above structural formula
can also be aromatic glyoxylic acid derivatives, for
example, the oximes of phenylglyoxylic acid, furangly-
, oxylic acid, 1,3-thiazoleglyoxylic acid, 1,3-oxazole-
-~ glyoxylic acid, and the substituted derivatives thereof,
for example, 2-amino-1,3-thiazoleglyoxylic acid oxime,
and the derivatives of the oximino function thereof,
~ 30 for example, the lower alkyl oxime derivatives such as
;
,1
i
1'.,
,
.
.
~ 17~66~
X-5155 -8-
the methoxyimino and ethoxylmino derivatives. Also, R
in the above formula represents the acyl portion of
phenylglycines wherein the amino group is substituted,
for example, by a carbamoyl group or a substituted
carbamoyl group, wherein the nitroge~ of the carbamoyl
group is substituted with an acyl moiety, for example,
acetyl, benzoyl, cinnamoyl, furoyl, thenoyl, and the
like. Also, the amino group of the phenylglycine can
be substituted with a cyclic urea function, for example,
¦ 10 the amino group can be substituted with a l-imidaz-
¦ alidone-2-carbonyl group or with a 1-~4-ethylpiperazine-
2,3-dione)carbonyl group.
In the above formula, Rl i~ hydrogen or a
carboxy-protecting group which is readily removed under
hydrolysis conditions or by hydrogenolysis. Such
I groups are well known in the cephalosporin art and
¦ include the readily cleaved ester groups for example,
benzyl, diphenylmethyl, p-nitrobenzyl, p-metho~yben~yl,
phenacyl, halogenated phenacyl, haloalkyl, for example,
J 20 iodomethyl, 2,2,2-trichloroethyl, and 2,2,2-tribromo-
ethyl; branched alkyl, alkenyl, and alkynyl esters, for
example, t-butyl, 3-methylbutene-1-3-yl, and isopentenyl.
When in the above formula, Rl is hydrogen,
the compounds of the invention can be converted to
pharmaceutically acceptable salts, for example, the
alkali metal and alkaline earth metal salts such as
the sodium salt, the potassium salt, the lithium salt,
and the calcium salt; amine salts such as those formed
with pharmaceutically acceptable amines such as procaine,
¦ 30 abietyl amine, the ethanolamines, such as monoethanol-
... . ...... .. .. ...... . ....... . .... . . .. . . ................. ..... ..
~ ~ .
6 6 8
X-5155 -9-
amine and diethanolamine, and like amine and metal
. salts, and the ammonium salt. The salts can be formu-
lated into pharmaceutically useful parenteral dosage
forms for administration of the antibiotics.
Preferred compounds of the invention are those
represented by the above general formula wherein R3
is a l-(lH-tetrazol-5-ylmethyl)-lH-tetrazol-S-yl group
wherein the NH group of the terminal tetrazole can be
substituted with Cl-C3 alkyl.
A further preferred group of compounds of
the invention are represented by the following
structural formula I.
H R2
.. R ~ ~
. 15 , ~ ~ ~ H2-S-R3
OOR1
, wherein R is hydrogen or an acyl group of the formula
` 20 Rl-C-, wherein Rl is Cl-C4 alkyl, Cl-C4 alkyl substi-
J tuted by halogen or cyano;
' or R is an aroyl or aralkanoyl group of the formula
, O
q ~.
~ 25 R--~CH2 ~C-
:~1 wherein R2 is phenyl or a mono substituted phenyl group
of the formula
;1'
',
~ ~ . . .. . . .
, . .
.
. . .
.
~ 1746B8
. X-5155 -10-
i
! wherein a is halogen, amino, hydroxy, Cl-C4
alkyl, Cl-C4 alkoxy, cyano, hydroxymethyl,
aminomethyl, carboxamido, carboxymethyi,
or Cl-C4 alkoxycarbonylmethyl;
or R2 is a di- or tri-substituted phenyl group of the
formula
a~
wherein a', a'', and a''' are independently
1 hydrogen, halogen, hydroxy, Cl-C~ alkyl,
or Cl-C4 alkoxy; and
n is 0 or 1;
or R is a heteroarylalkanoyl group of the formula
O
R3-CH2-C-, wherein R3 is
~
1 H
1 25 b/ \ ~ b/ \ ~ HN/ ~ NH
j wherein each b is amino, protected amino,
~ 30 Cl-C3 alkyl or phenyl;
:.
' :
~ 174ii66~
X-5155 -11-
or R is an aryloxyacetyl or arylthioacetyl group ofthe formula
2 ..
S~ ~Zt--CH2-c-
wherein R2 has the same meanings as defined
I above and z is O or S;
or R is an aralkanoyl or heteroarylalkanoyl substituted
~ group of the formula O
¦ 10 R4-CH-C-
, Q
wherein R4 is R2, as defined above and in
addition is thienyl, furyl, or 1,4-cyclo-
15hexadienyl; Q i9 hydroxy, formyloxy, carboxy,
the sulfo group -SO3H, or amino;
or R is an oximino-substituted aralkanoyl or hetero-
arylalkanoyl group of the formula
20R5-C-C-
OR
'
wherein R5 is R2 and R3 as each is defined
25above, and R6 is hydrogen or Cl-C3 alkyl;
or R is a group of the formula
~ 4 "
;i R -CH-C-
i NH
il c=o
.~ 30R7
i
. ~ ,,
., .~
. . . .
J
,
l 17466~
X-5155 -12-
wherein R7 is hydroxyphenyl or hydroxypyridyl
or a group of the formula
O
R3-C_N_
CH3
!
wherein R8 is Cl-C4 alkylamino, phenyl, furyl,
! styryl, nitrostyryl, or chlorostyryl; or
:, R7 is a group of the formula
R~
! (CHz)
m
wherein n' is 1 or 2 and m is 2 or 3 with
~ the limitation that when n' is 2, m is 2; and
s R9 is hydrogen, Cl-C3 alkyl, or Cl-C3 alkyl-
sulfonyl;
' Rl is hydrogen or a carboxy protecting group;
R2 is hydrogen or methoxy;
R3 is a bis-tetrazolyl methyl group of the formula
1, ~
~, 25 ~H ~ or ~ ~ Hz~
-I s
':`I
wherein R4 is hydrogen, or Cl-C3 alkyl; and when Rl is
hydrogen the pharmaceutically acceptable salts thereof.
:
. :
. . ' :
- .' .
.
. .
: ~ .
1 174568
X-5155 -13-
Examples of the foregoing preferred compounds
are described below in Table 1.
TABLE 1
5Bis-Tetrazolemethyl Cephalosporins
H R2
R ~ t
~ CH2-S-R~
OOR
R = Rl
R Rl R2 Rl (R4)
15 acetyl H H A H
; acetyl Na OCH3 A CH3
acetyl H H B CH3
propionyl H H A H
butyryl H H A H
~,20 chloroacetyl H H A H
. chloroacetyl H OCH3 A CH3
chloroacetyl H H B H
cyanoacetyl H H A H
cyanoacetyl H OCH3 A H
.25 cyanoacetyl H H B CH3
., ~
`I R = R8--(CH2)n-~--
j (n = o)
, 30 benzoyl H H A H
benzoyl H H B H
benzoyl H H A CH3
I . . . .... .. .. .. . .
. ~ . - ~, . .
,
~ , :
~ ~;
1 1746S8
X-5155 -14-
Table 1 cont.
R Rl R2 Rl (R4)
p-chlorobenzoyl H H A CH3
2,6-dimethoxy- H H A H
benzoyl
p-hydroxybenzoyl H H A H
p-toluyl H H A H
p-anisoyl H H B H
10 -m-cyanobenzoyl H H B CH3
p-methoxycarbonyl- H H A H
benzoyl
., ~
R = R2--( CH2 ) n~ l_
(n = 1)
phenylacetyl H H A H
phenylacetyl H OCH3 A CH3
phenylacetyl H H B H
p-chlorophenyl- H H A H
acetyl
p-methylphenyl- H H B H
acetyl
3,4-dimethyl- H H A H
phenylacetyl
o-æminomethyl- H H A CH3
phenylacetyl
o-aminomethyl- H H B H
phenylacetyl
p-hydroxymethyl- H H A H
phenylacetyl
p-hydroxymethyl- H H A H
' 30 phenylacetyl
p-carboxamido- H H A H
phenylacetyl
. ~
1 174688
X-5155 -15-
Table 1 cont.
R Rl R2 Rl (R4)
p-methoxyphenyl- Na H A H
acetyl
p-methoxyphenyl- H OCH3 B CH3
acetyl
3,4-dimethoxy- H H A H
phenylacetyl
10 p-cyanophenylacetyl H H A CH3
p-ethoxyphenyl- H H B H
acetyl
p-isopropoxy- H H A CH3
phenylacetyl
3-ethylphenylacetyl H H A CH3
15 2,6-dimethyl- H H A CH3
; phenylacetyl
2,6-dimethoxy- H OCH3 A CH3
phenylacetyl
p-bromophenylacetyl H H B H
~ m-fluorophenyl- H H A H
;, 20 acetyl
'. 3,4-dihydroxy- H H A CH3
; phenylacetyl
3-chloro-4-hydroxy- H H A H
phenylacetyl
3,5-dichloro-4- H H ~ H
hydroxyphenylacetyl
~; 3-methoxy-4- H H A CH3
hydroxyphenylacetyl
: 3-methyl-4-hydroxy- H H B H
phenylacetyl
o-ethoxyphenyl- H OCH3 A H
i 30 acetyl
~1 p-fluorophenyl- H H A H
:: acetyl
i
~ .. . . . ~ . _ . _.. _ .. ~ _ .. _ .. _ .. _ _ . .. _ . .... .. _ _ ~ .. . . _ _ _ _._ . _ . _ . ~ . . .. .
.
. ,~ ,. . .
-
1 174668
, X-5155 -16-
;
Table 1 cont.
R Rl R2 R3 (R4)
_
p-carboxyphenyl- H H H CH3
acetyl
3-bromo-4-methoxy- H H B H
phenylacetyl
p-t-butylphenyl- H H A CH3
acetyl
R = R3{~H~--
2-thienylacetyl H H A H
2-thienylacetyl H OCH3 A E
2-thienylacetyl pNB H B H
15 3-thienylacetyl H H A H
2-furylacetyl H H A CH3
2-furylacetyl H OCE3 B H
lH-tetrazol-l- H H A CE3
i ylacetyl
' lH-tetrazol-l- H OCH3 A CH3
:! 20 ylacetyl
, lH-tetrazole-l- H H B H
ylacetyl
! 2H-tetrazol-5- H H A H
ylacetyl
2H-tetrazol-5- H OCH3 A H
2S ylacetyl
2H-tetrazol-5- H OCH3 B CH3
ylacetyl
1,3-thiazol-4- H ~ A H
acetyl
1,3-oxazol-4- H H A CE3
~ 30 ylacetyl
.. ....
.
.
.
.
,
. . .
1 174668
X-5155 -17-
Table 1 cont.
R Rl R2 R3 (R4)
2-methyl-1,3- H H B H
thiazol-4-ylacetyl
2-phenyl-1,3- H OCH3 A CH3
thiazol-4-ylacetyl
2-phenyl-1,3- H OCH3 B CH
oxazol-4-ylacetyl 3
2-amino-1,3- H H A H
thiazol-4-ylacetyl
2-amino-1,3- Na H A CH
thiazol-4-ylacetyl 3
2-amino-1,3- H OCH3 A H
thiazol-4-ylacetyl
2-amino-1,3- H H B H
thiazol-4-ylacetyl
R
R = R2-(Z)-CH2~-
phenoxyacetyl H H A H
20 phenoxyacetyl H H A CH3
phenoxyacetyl H H B H
phenoxyacetyl H OCH3 A H
p-chlorophenoxy- H H A H
acetyl
3,4-dichloro- H H ~ H
`! 25phenoxyacetyl
: t p-methylphenoxy- H OCH3 A CH3
~ 1 acetyl
:~ p-fluorophenoxy- H H A H
acetyl
~ p-methoxyphenoxy- H H ~ H
:, 30 acetyl
;
.
,:
'
1 174668
X-5155 -18-
Table 1 cont.
R Rl R2 Rl (R4)
p-hydroxyphenoxy- H OCH3 A H
acetyl
3-chloro-4-methoxy- H H B H
phenoxyacetyl
: 3,5-dichloro-4- H H A CH
hydroxyphenoxyacetyl 3
phenylthioacetyl H H A H
10 phenylthioacetyl H H B H
phenylthioacetyl H OCH3 A H
p-chlorophenyl- H H A H
thioacetyl
3,4-dichlorophenyl- H H B H
thioacetyl
15 3,5-dichlorophenyl- H H A CH3
thioacetyl
:p-fluorophenylthio- H H A H
;acetyl
,p-methylphenylthio- H H ~ H
;acetyl
3-hydroxy-4-methyl- H H A H
phenylthioacetyl
.p-ethoxyphenylthio- H OCH3 A H
acetyl
`: 8
R = R
Q
i; phenylglycyl H H A H
phenylglycyl H OCH3 A H
~ p-hydroxyphenyl- H H A H
i 30 glycyl
~ 3-chloro-4-hydroxy- H H A H
.:l phenylglycyl
.
'~ '
. j
~'
. :
' ; , , ~
..
1 174668
X-5155 -19-
Table 1 cont.
R Rl R2 R3 (R4)
_
3,5-dichloro-4- H H A H
hydroxyphenyl-
glycyl
: mandeloyl H H A H
mandeloyl H H B H
mandeloyl H OCH3 A CH3
a-carboxyphenyl- H H A H
acetyl
. a-carboxyphenyl- H H B H
acetyl
a-carboxyphenyl- H OCH3 A H
acetyl
a-carboxy-4- H OCH3 A H
hydroxyphenyl-
acetyl
-carboxy-4- H OCH3 B H
~ hydroxyyhenyl-
i acetyl
a-carboxy-4- H OCH3 B CH3
hydroxyphenyl-
acetyl
a-sulfophenylacetyl H H A H
a-sulfophenylacetyl H H B H
a-sulfophenylacetyl H OCH3 A H
a-amino-2-thienyl- H H A H
acetyl
a-amino-2-thienyl- H H B H
acetyl
a-amino-2-furyl- H a A CX3
acetyl
: I .
~ 30
I
. ` ,: , :
.
,
,
.
' ~ . .
1 17466~
X-5155 -20-
Table 1 cont.
R Rl R2 Rl (R4)
R = Rs~ ~_
oR6
a-hydroximino H H A H
10phenylacetyl
a-hydroximino- H H B H
phenylacetyl
a-methoximino- H H A H
phenylacetyl
a-hydroximino-2- H H A H
: lSfurylacetyl
a-hydroximino-2- H OCH A H
furylacetyl 3
a-methoximino-2- H H A H
furylacetyl
: a-methoximino-2- H H A H
thienylacetyl
a-hydroximino-(2- H H A H
amino-1,3-thiazol-
4-yl)acetyl
a-hydroximino-(2- Na H A CH3
amino-1,3-thiazol-
4-yl)acetyl
: 25 a-hydroximino-( 2- H H B H
. amino-1,3-thiazol-
, 4-yl)acetyl
~, a-hydroximino-(2- H OCH A H
; amino-1,3-thiazol- 3
~l 4-yl)acetyl
:, 30 a-methoximino-(2- H H A H
` amino-1,3-thiazol-
.~ 4-yl)acetyl
:,'
, ~ .
;
,'
~. .. , .. ... . _ . _ .. __ . _ .. _ . ._ _. .. _. .. _ ._ .. .. . . .. ._ .. _ _ .
:. ' ' ~ , :
;
. .
,
. , : .
1 174~
X-5155 -21-
Table 1 cont.
R Rl R2 Rl (R4)
a-methoximimo-(2- H H A CH3
amino-1,3-thiazol-
4-yl)acetyl
a-methoximino-(2- H H B H
amino-1,3-thiazol-
4-yl)acetyl
a-methoximino-(2- H OCH3 A H
amino-1,3-thiazol-
4-yl)acetyl
.,
R = R4~
I¦IH
a-~4-hydroxybenz- H H A H
amido)phenylacetyl
a-(2-hydroxybenz- H H A H
amino)phenylacetyl
20 a-(2-hydroxybenz- H H A CE3
amido)-2-thienyl-
acetyl
a-(2,3-dihydroxy- H H A H
benzamido)phenyl-
acetyl
.25 a-(4-hydroxybenz- H H A H
iamido)-4-hydroxy-
phenylacetyl
i a-(4-hydroxy- H H A CH3
-I pyridine-3-car-
boxamido)phenyl-
acetyl
.
,
:
.
.
i
. . .
1 174~68
X-5155 --22--
Table 1 cont.
R Rl R2 R3 (R4)
a-(4-hydroxy- H H B H
pyridine-3-car-
boxamido)phenyl-
acetyl
a-(4-hydroxy- H H A H
pyridine-3-car-
boxamido)phenyl-
acetyl
a-(3-methylcar- H H A H
' bamoyl-3-methyl-1-
ureido)-a-phenyl-
acetyl
a-(3-methylcar- H H A H
bamoyl-3-methyl-
1-ureido)-a-(4-
hydroxyphenyl)-
I acetyl
a-(3-benzoyl-3- H H A CH3
¦ methyl-l-ureido)-
a-phenylacetyl
a-[3-to-chloro- H H A H
1 20 benzoyl)-3-methyl-
I l-ureido}-a-(2-
thienyl)acetyl
, a-~3-(2-furoyl)-3- H H B H
-, methyl-l-ureido]-
~' a-phenylacetyl
i a-~3-(2-thienoyl)- H H A H
3-methyl-1-ureido]-
a-phenylacetyl
1 a-(3-cinnamoyl-3- H OCH3 A H
methyl-l-ureido~-
, a-phenylacetyl
a-(3-cinnamoyl-3- H H A H
i 30 methyl-l-ureido)-
`l a-phenylacetyl
i
~. i :
.. ,
: i
,~ j
, . . . . . . ... . . . .
.. ..
'
: .
; . :
. ..
...... . . .
, :
1 174~88
X-5iS5 -23-
Table 1 cont.
R Rl R2 R3 (R4)
a-13-(4-nitrocinna- H H A CH
moyl)-3-methyl-1- 3
ureido]-a-phenylacetyl
a-[3-(4-nitrocinna- Na H B H
moyl)-3-methyl-1-
ureido]-a-phenylacetyl
: a-[3-(4-chlorocin- H H A H
namoyl)-3-methyl-1-
ureidol-a-phenylacetyl
: a-(imidazolidin-2- H H A H
: one-l-ylcarbonyl-
amino)-a-phenyl-
acetyl
a-(imidazolidin-2- H H A CH
one-l-ylcarbonyl- 3
! 1S amino)-a-(4-hydroxy-
phenyl)acetyl
a-(3-acetylimida- H . H A H
zolidin-2-one-1-yl-
carbonylamino)-a-
¦ phenylacetyl
20 a-(3-acetylimida- H H B H
zolidin-2-one-1-
yl-carbonylamino)-
a-phenylacetyl
a-(3-methylsul- H H A H
fonylimidazolidin-
2-one-1-ylcar-
bonylamino)-a-phenyl-
acetyl
a-(3-methylsul- H H B H
7 fonylimidazolidin-
2-one-1-ylcarbonyl-
amino)-a-phenylacetyl
a-(3-methylsul- H H A CH3
l 30 fonylimidazolidin-
. 2-one-1-ylcarbonyl-
amino)-x-phenylacetyl
'~
' 1 '
.. .
! i,
.
.. ~ ' ,
. ~ .
~' ' . , ' . .
.
'
: '
'
~ 174558
X-5155 -24-
Table 1 cont.
R Rl R2 R3 (R4)
a-(4-ethylpiper- H H A H
azin-2,3-dione-1-
ylcarbonylamino)-
a-phenylacetyl
a-(4-ethylpiper- H H B H
azin-2,3-dione-1-
ylcarbonylamino)-
a-phenylacetyl
' a-(4-ethylpiper- H H B CH3
- azin-2,3-dione-1-
: ylcarbonylamino)-
a-phenylacetyl
a-(4-ethylpiper- H H A H
azin-2,3-dione-1-
ylcarbonylamino)-
a-(4-hydroxyphenyl)-
acetyl
a-(4-ethylpiper- H H A CH3
. azin-2,3-dione-1-
: ylcarbonylamino)-
; a-(4-hydroxyphenyl)-
acetyl
-(4-methylpiper- H H A H
azin-2,3-dione-1-
: ylcarbonylamino)-
a-(2-thienyl)acetyl
a-(piperazin-2,3- H H A H.
dione-l-ylcarbonyl-
amino)-a-phenyl-
acetyl
1/ A = ~ ~ ~ = ~ H~ N~
~~
. . .
.
1 17468~
X-5155 -25-
The compounds of this invention have the
normal stereochemistry of the cephalosporin antibiotics
with the 7-position side chain, R, having the ~-con-
figuration while the term "R2", the 7-methoxy group,
has the a-configuration.
The compounds of the invention, represented
by the above formula wherein R is an acyl group, are
prepared by the N-acylation of a 7-amino-3-(bis-tetra-
zolmethyl) compound of the formula I wherein R is H.
The 7-amino nucleus compounds are represented by the
following structural formulas II and III.
R2
~ ~ ~
OOR~ H2 ~ II
~!
Rz
H2N~/S\t
H2-S ~ ~ H2~
OOR1 4
`' wherein Rl, R2, and R4 have the same meanings as defined3 above.
The N-acylation of the above 7-amino nucleus
` 30 compounds can be carried out by a wide variety of
;',
.~ .
:- . .. . .
1 17a~SS8
X-SlSS -26-
acylation methods commonly employed in the cephalosporin
art. For example, the acylation can be carried out
under non-aqueous condltions or under aqueous conditions.
Under the former conditions, an organic soluble active
derivative of the carboxylic acid used to form the
desired acyl group is formed and used to acylate the
7-amino nucleus compound. Examples of active deriva-
tives of the carboxylic acid which can be formed include
the active esters, for example those formed with
hydroxybenzotriazole, or N-hydroxysuccinimide; mixed
anhydrides formed with the carboxylic acid and methyl
chloroformate or isobutyl chloroformate; and acid
; azides. The free acids themselves can be employed
when used in the presence of a condensing agent such
as EEDQ. Acylation can also be carried out under
aqueous conditions, for example by employing an acid
; halide, for example acid chloride or acid bromide in
1 an aqueous organic medium containing a hydrogen halide
acceptor, for example a tertiary alkyl amine such as
triethylamine or pyridine or, alternatively, a base
: such as sodium bicarbonate or sodium carbonate. In
~; general, higher yields are obtained in acylations
. ~
carried out under non-aqueous conditions.
` The 7-amino-3-~bis-tetrazolmethyl) nucleus
` 25 compounds of the above formulas are prepared with
7-aminocephalosporanic acid ~7-ACA). In preparing
these nucleus compounds, 7-aminocephalosporanic acid
or an ester thereof is reacted with the l-~lH-tetrazol-
5-ylmethyl)-lH-tetrazol-5-thiol or the 2-~lH-tetrazol-
, 30 5-ylmethyl)-2H-tetrazol-5-thiol to form the compounds
.~ I
~'
_ .... .. ~ . , .. _ .. _ . _ _ . _ _ _ _ _ . .. ... _ . . _ .. . .. .. _ _ . _ _ . _ . ,
- .
,
.
:~ :
6 6 ~
X-5155 -27-
of the above formulas. The reaction, which results in
the replacement of the acetoxy group in the 3-position
; of 7-ACA with the bis-tetrazolmethyl thiol, can be
carried out under non-aqueous conditions when in the
above formulas Rl is hydrogen or an ester, and under
aqueous conditions when Rl is hydrogen.
The preparation of the nucleus compounds
; represented by the above fo~mula is carried out under
non-aqueous conditions as follows. 7-Aminocephalo-
10 sporanic acid is reacted with the bis-tetrazolmethyl
s thiol at a emperature between about 25C. to about
80C. in glacial acetic acid in the presence of boron
trifluoride etherate. The reaction proceeds at a
convenient rate at a temperature of about 60-80C. and
15 higher yields are obtained when an excess of the thiol
is employed. Following the reaction, the mixture is
diluted with water and the pH is adjusted to the iso-
, electric point of the substituted nucleus free acid.
I At the isoelectric point of about pH 4 0-5.0, the
1 20 product precipitates from the reaction mixture.
j The displacement under aqueous conditions is
carried out as follows. An aqueous solution of 7-ACA
at a basic pH of about pH 7.0-9.0 is mixed with a
solution of the bis-tetrazolmethyl thiol, in the form
25 of the sodium or potassium salt, and the reaction
mixture is stirred with mild heating. The reaction
product mixture is acidified with a mineral acid such
! as hydrochloric acid to the isoelectric point. The
~¦ 3-substituted 7-amino free acid compound precipitates
from the reaction mixture nd is recovered by filtration.
i __ _ _ __ _, ..... _ .
1 t74~6~
X-5155 -28-
The substituted nucleus compound can be purified if
necessary by recrystallization from a suitable solvent
or, alternativeiy, via a salt form thereof such as the
hydrochloride salt or the tosylate salt.
The 3-substituted 7-amino nucleus co~pounds
represented by the above formulas are valuable inter-
mediates useful in the preparation of the antibiotic
compounds of this invention. As mentioned above, the
7-amino group of the nucleus compound can be acylated
with the desired carboxylic acid and preferably an
active derivative thereof. The N-acylation of the
above 7-amino substituted nucleus compounds is a coupling
; reaction of a carboxylic acid and an amino compound and
can be carried out by acylation methods employed in the
acylation of the 7-aminocephalosporin nucleus compound,
for example, 7-ACA and 7-ADCA. To illustrate these
acylation methods in the preparation of the compounds
of this invention, the following paragraphs describe
various acylation methods which are used in the prepara-
! 20 tion of the compounds of the invention.
Aqueous Acylation
The compounds of the above formulas II and IIIcan be acylated under aqueous conditions under essen-
' 25 tially Schotten-Bauman conditions, for example, an acid
~, halide such as the acid chloride is reacted in aqueous
¦ acetone with the 3-substituted 7-amino nucleus compound
in the form of a sa't in the presence of a hydrogen
halide accepting base. The base can be an inorganic
base such as sodium or potassium carbonate or bicarbonate
l or a tertiary amine such as pyridine or a trialkylamine,
,,
;1
.: ~
,~' ;'` , .
~ ` . . :
,` :
,
117466~
X-5155 -29-
such as trimethylamine or triethylamine. The acylation
is generally carried out at room temperature and the
product is readily recovered. For example, when a salt
form of the 7-amino nucleus compound is employed the
reaction mixture is acidified to convert the N-acylated
nucleus to the free acid which is extracted with a
water immiscible organic solvent.
The acyl chlorides used in the acylation are
; best prepared by reacting the free acid with oxalyl
chloride in an inert organic solvent in the presence
of a hydrogen chloride acceptor such as propylene
i oxide. Preferably, the oxalyl chloride is reacted with
the acid in the organic solvent in the presence of a
catalytic amount of dimethylformamide. Solvents such
as THF and acetonitrile are suitable.
Examples of acid chlorides which can be used
to acylate the 3-substltuted 7-amino nucleus compounds
(Formula I, R~H) to provide the antibiotics of the
Formula I are acetyl chloride, cyanoacetyl chloride,
chloroacetyl chloride, propionyl chloride, benzoyl
chloxide, phenylacetyl chloride, p-chlorophenylacetyl
chloride, p-hydroxyphenylacetyl chloride, p-methoxy-
phenylacetyl chloxide, o-aminomethylphenylacetyl chloride
hydrochloride, phenoxyacetyl chloride, p-chlorophenoxy-
acetyl chloride, p-methoxyphenoxyacetyl chloride,
~ phenylmercaptoacetyl chloride, 3,5-dichlorophenylmercapto-
¦ acetyl chloride, p-fluorophenylmercaptoacetyl chloride,
thiophene-2-acetyl chloride, thiophene-3-acetyl chloride,
¦ furan-2-acetyl chloride, lH-tetrazole-l-ylacetyl chlo-
ride, lH-tetrazole-5-ylacetyl chloride, mandeloyl
1 ~746B8
X-5155 _30_
chloride, o-formylmandeloyl chloride, -(p-nitrobenzyl-
oxycarbonyl)phenylacetyl chloride, a-sulfophenylacetyl
ch}oride and, a-(diphenylmethyloxycarbonyl)-2-thienyl-
acetyl chloride.
In an example of the aqueous acylation a
solution of 7-amino-3-[1-(lH-tetrazole-5-ylmethyl)-lH-
tetrazole-5-ylthiomethyl]-3-cephem-4-carboxylic acid
(formula 2, Rl=R2=R4=H) in aqueous acetone containing
a slight excess of sodium carbonate is treated with
stirring at a temperature between about -5C. and 35C.
with a solution of a slight molar excess of thiophene-2-
acetyl chloride in acetone. The acylation product,
7-(2-thienylacetamido)-3-[l-(lH-tetrazole-5-ylmethyl)-
lH-tetrazole-5-ylthiomethyl]-3-cephem-4-carboxylic
acid represanted by the following formula is obtained.
.1 ~
H
The 3-substituted 7-amino nucleus compounds
, 25 can also be acylated under aqueous conditions with
¦ active esters of carboxylic acids formed with hydroxy-
¦ benzotriazole or hydroxysuccinimide. For example,
syn 7~-[a-methoximino-a-(2-amino-1,3-thiazol-4-yl)-
acetamido]-3-~2-(lH-tetrazole-5-ylmethyl)-2H-tetrazol-
5-ylthiomethyl]-3-cephem-4-carboxylic ~cid is prepared by
~ 174868
X-5155 -31-
acylating 7-amino-3-[2-(lH-tetrazole-5-ylmethyl)-2H-
tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylic acid of
the formula
S HZN~T'S`T =\1
~CI 12--SY~ I~H2~
OOH H
with a-methoximino-a-(2-amino-1,3-thiazol-4-yl)acetic
; acid hydroxybenzotriazole ester of the formula
H2N- ~
~ OCH3
wherein BT - benzotriazol-l-yl
The acylation is carried out in aqueous acetone at a
pH of about 7-8.
~on-Aqueous Acylation
The acylation of the 3-substituted 7-amino
nucleus compounds is carried out under non-aqueous
conditions with an active derivative of the carboxylic
acid. Generally, the carboxy group of the nucleus
is protected during the acylation, for example, with
a carboxylic acid protecting group. Suitable protecting
groups include, for example, p-nitrobenzyl, p-methoxy-
benzyl, benzyl, diphenylmethyl, 2,2,2-trichloroethyl,
t-butyl, and like ester groups which are easily cleaved
under hydrolytic or hydrogenolytic conditions.
1 ~ 745~a
X-5155 -32-
Alternatively, the carboxy group of the 7-amino nucleus
compound can be temporarily protected during the
N-acylation as a silyl ester, for example, the trimethyl-
silyl ester formed by reacting a suspension of the freenucleus acid with a silylating agent such as trimethyl-
silyl acetamide or bis-trimethylsilyl acetamide.
Active derivatives of the carboxylic acid
acyl moiety which can be employed in the acylation
include, for example, the pentachlorophenyl ester, a
mixed anhydride, for example, the mixed anhydride
formed with methyl chloroformate or with isobutyl
chloroformate; an active ester formed with N-hydroxy-
succinimide or hydroxybenzotriazole (HBT); an acid
halide or an acid azide.
The free acid itself can be coupled with the
7-amino nucleus compound in the presence of a condens-
ing agent, or example, a carbodiimide such as dicyclo-
hexylcarbodiimide.
The following illustrate the non-aqueous
acylation method for the preparation of compounds of
the formula I.
7-Amino-3-[1-(lH-tetrazol-5-yl-methyl)-iH-
tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylic acid is
reacted in acetonitrile with trimethylsilyl acetamide
to form the trimethylsilyl ester of the 7-amino nucleus,
. and the nucleus ester is acylated.with the mixed anhydride
of an amino-protected phenylglycine formed with methyl
chloroformate to provide the amino-protected 7-phenyl-
acetamido-3-[1-(lH-tetrazol-5-ylmethyl)-lH-tetrazol-5-
ylthiomethyl]-3-cephem-4-carboxylic acid trimethylsilyl
`: .' ~. ~ ' ,
.
'. ' ' '
.
~ . :
-
1 174~68
X-5155 _33_
ester. The trimethylsilyl ester is hydrolyzed to the
free acid and the amino protecting group is removed
to provide the deprotected amino acid. ~he above is
illustrated by the following reaction scheme wherein
the t-butyloxycarbonyl group is the amino protecting
group.
l0 COOSi(CHa)~
i(CH3)3
~ H--C~HD
: t-30C
CH~ f `LH2-S ~
1~ 0 t ; ~ N
t-~OC ~OOSi(CH3)3 ~Hz - ~
¦ H ~i~CH3)3
~I H20
r ~ N
~t~
: 30
_. . . . . _ . _ _ _ _ _ _ _
,:
.
1 174~68
X-5155 _34_
The t-butyloxycarbonyl group (t-BOC) and the
trimethylsilyl ester group are removed upon treatment
of the protected intermediate under acid hydrolysis
conditions.
~y~ 7~-ca-Methoximino-a-(2-amino-l~3-
thiazol-4-yl)acetamido]-3-[2-(lH-tetrazol-5-ylmethyl)-
2H-tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylic acid
is prepared by acylating 7-amino-3-[2-(lH-tetrazol-S-
ylmethyl)-2H-tetrazol-5-ylthiomethyl]-3-cephem-4-
carboxylic acid trimethylsilyl ester with the hydroxy-
benzotriazole ester of a-methoximino-a-(2-amino-1,3-
tria~ol-4-yl)acetic acid in a dry solvent such as
tetrahydrofuran or acetonitrile. The produ~t is re-
covered from the reaction mixture by extraction follow-
ing acid hydrolysis of the trimethylsilyl ester groupand is purified by reversed phase silica gel HPLC.
~ he compounds represented by the formula I,
wherein R is an acyl group represented by the formula
R4~ ~ -
~H
wherein R is hydroxyphenyl or hydroxypyridyl, are
: 25 prepared by acylating the a-amino group of a 7-phenyl-
glycyl, thienylglycyl, or furylglycyl substituted
cephalosporin represented by the formula I wherein R
is an acyl group of the formula
. _ . . , . .... .... _ ... _ _ _ . _ _ ... . _ .. . . _ . . _ _ . _ _ .. . . .... _ _ _ . _ . _ .. _ _ . . . .
..
.
.
~, . .. .
' . . ,' ~' ~,
:
- . ~
, . ~. .
1 174~88
X-5155 -35-
R4~ ~ -
~;
wherein Q is amino, with an active ester of the hydroxy
substituted benzoic acid or the hydroxy substituted
pyridine carboxylic acid. Preferably, the acylation
is carried out by first preparing the hydroxybenzo-
triazole ester of the acid in the presence of a
condensing agent such as a carbodiimide, for example,
dicyclohexyl carbodiimide. The acylation of the phenyl-
glycyl substituted cephalosporin of the formula I is
carried out under non-aqueous conditions, for example,
in dry THF or acetonitrile.
Representative of the hydroxy substituted
benzoic acids and the hydroxy substituted pyridine
carboxylic acids which can be employed are 4-hydroxy-
benzoic acid, 3-hydroxybenzoic acid, 2-hydroxybenzoic
acid, 2,3-dihydroxybenzoic acid, 2,6-dihydroxybenzoic
acid, and 3,4-dihydroxybenzoic acid, 3-hydroxypyridine-
4-carboxylic acid, 2,3-dihydroxypyridine-4-carboxylic
acid, 4-hydroxypyridine-3-carboxylic acid, 2~4-
dihydroxypyridine-3-carboxylic acid, and 4,5-dihydroxy-
pyridine-3-carboxylic acid.
An example of the preparation of the above
hydroxy-substituted benzoic ~nd hydroxy-substituted
pyridine carboxylic acid derivatives is illustrated
in the following reaction scheme in which 7-phenyl-
glycylamido-3-[l-(lH-tetrazol-5-ylmethyl)-lH-tetrazol-
5-ylthiomethyl]-3-cephem-4-carboxylic acid is reacted
in acetonitrile with 2-hydroxybenzoic acid hydroxy-
.
: `
-~..... :: . -
: .,:
.
1 174~
X-5155 -36-
benzotriazole ester to provide 7-[a-(2-hydroxybenz-
amido)phenylacetamido]-3-[1-(lH-tetrazol-5-ylmethyl)-
lH-tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylic acid.
,s~ ~5~
H
~ ~COOBT
~1,
\e~ CH5~--5
2 0 H~
~-
The compounds represented by the formula I
wherein R7 is a group of the formula
,, Ra_~
H~
~ 30
:
~.
. ~, , .
., j , :.
,~
.
: ' ~
1 174BB8
X-5155 -37-
are prepared with a compound of the formula I wherein
R is a phenylglycyl, furylglycyl, or thienylglycyl
group represented by the term R -CH~Q)-C=O- wherein Q
is amino. When in the above formula R8 is phenyl,
chlorophenyl, furyl, styryl, nitrostyryl, or chloro-
styryl, the compounds are prepared by acylating the
a-amino group of the phenylglycyl substituted cephalo-
sporin with an N-chlorocarbonyl amide derivative repre-
sented by the following formula.
R~ CI
H3
The N-chlorocarbonyl amides are prepared by
reacting the N-methyl amide of the R8-COOH acid with
phosgene in an inert solvent in the presence of a
hydrogen halide acceptor. Representative of the
N-methyl amide9 which can be employed in the acylation
are N-methylbenzamide, N-methyl-4-chlorobenzamide, N-
methyl-3-chlorobenzamide, N-methyl-2-furoic acid amide,
N-methyl-3-furoic acid amide, N-methylcinnamide, N-
methyl-4-chlorocinnamide, N-methyl-4-nitrocinnamide,
N-methyl-2-chlorocinnamide, and N-methyl-2-nitrocinnamide.
The N-methyl amide is reacted with phosgene
in an inert solvent such as a chlorinated hydrocarbon
solvent, for example, methylene chloride or trichloroethane
in the presence of a hydrogen halide acceptor, for
example, a tri-lower alkyl amine, for example, tri-
ethylamine or pyridine.
1 17~6~
X-5155 -38-
The N-chlorocarbonyl amides are coupled with
the a-amino group of the cephalosporin compound via an
N-acylation which can be carried out under aqueous or
non-aqueous conditions in the presence of a hydrogen
halide acceptor. The acylation is carried out at a
temperature between about -5C. and about 35C. and
preferably at about 0-5C. The solvents which can be
employed in the acylation include acetonitrile, TXF,
DMF, and dimethylacetamide. In an example of the
acylation, 7-phenylglycylamido-3-[1-(1-methyl-lH-
tetrazol-5-ylmethyl)-lH-tetrazol-5-ylthiomethyl]-3-
cephem-4-carboxylic acid is converted to the trimethyl-
silyl ester in acetonitrile with trimethylsilyl
acetamide, and the ester is reacted with N-chloro-
}5 carbonyl-N-methyl-2-chlorobenzamide at a temperature
of about 20C. to provide the compound of the formula I,
7-~a-(N-methyl-2-chlorobenzamido)phenylacetamido]-3-[1-
(l-methyl-lH-tetrazol-5-ylmethyl)-lH-tetrazol-5-ylthio-
methyl]-3-cephem-4-carboxylic acid trimethylsilyl ester.
The ester is hydrolyzed with mild acid hydrolysis following
the acylation and the free acid is recovered by extraction
from the reaction mixture.
The compounds represented by the formula I
wherein R8 is a Cl-C4 alkylamino group are likewise
prepared by the acylation of the -amino substituted
cephalosporin represented by the formula I with an
N-alkyl-N'-methyl-N'-chlorocarbonyl substituted urea.
For example, N,N'-dimethylurea is reacted with phosgene
in an inert solvent to provide the ~-chlorocarbonyl
derivative which is then used in the acylation of the
. . .
.... . . ....
.
. ' ', ~ ~
.: , ~ ', ~, '.
: . . , :,
1 174668
X-5155 -39-
a-amino substituted cephalosporin under the acylation
conditions described above. Representative of the
N,N'-disubstituted ureas which can be used in the
acylation to prepare compounds of the formula I are
N,N'-dimethylurea, N-ethyl, N'-methylurea, N-(n-butyl)-
N'-methylurea and like N-(Cl-C4 alkyl-N'-methylureas.
Another process for preparing the compounds
of this invention is to react a 7-acylamino-3-acetoxy-
methyl-3-cephem-4-carboxylic acid compound represented
by the formula
H / \
~s~ ~t--CH2--O~-CH3
~OOH
wherein R is an acyl group as defined for formula I,
with a bis-tetrazolemethyl thiol compound represented
by the formula
~-CHa~ V
wherein R4 is hydrogen or Cl-C3 alkyl, to form the com-
pound of formula I. The reaction can be carried out at
: a temperature between about 20C and about 75C under
aqueous conditions at a pH of about 7 to about 9 and
preferably in the presence of a water miscible organic
solvent such as acetonitrile or tetrahydrofuran.
'
.
1 174~6~
X-5155 _40-
The above reaction can also be carried ou~
under non-aqueous conditions by following the process
described by Hatfield in U.S. Patent No. 4,144,391.
The compounds represented by the formula I
wherein R2 is methoxy are prepared either by the acyla-
tion of the corresponding 7-amino-7-methoxy compound
(R=hydrogen~, or by the replacement of the 3-acetoxy
group of a 7-acylamino-7-methoxycephalosporanic acid,
or by the replacement of a 3' halo group of a 7-acyl-
amino-7-methoxy-3-halomethyl-3-cephem-4-carboxylic acid
ester.
Acylations of the 7-amino-7-methoxy 3-sub-
stituted nucleus compounds are carried out with nucleus
in an esterified form, and preferably under non-aqueous
acylation conditions. The non-aqueous acylation condi-
tions described hereinabove are suitable acylationmethods for the 7-methoxy nucleus esters.
~ lternatively, a 7-acylamino-7-methoxy-3-
acetoxymethyl-3-cephem-4-carboxylic acid can be reacted
with a bis-tetrazolmethyl thiol to prepare a compound
of the invention by following the reaction procedures
described above for the non-methoxylated cephalosporin
compounds.
In yet another alternative for preparing the
7-methoxycephalosporin compounds of the formula I,
a 7-acylamino-7-methoxy-3-bromomethyl-3-cephem-4-
carboxylic acid ester is reacted with a bis-tetrazol-
methyl thiol to provide the compound of the invention.
For example, p-nitrobenzyl 7-(2-thienylacetamido)-
7-methoxy-3-bromomethyl-3-cephem-4-carboxylate is
.
' '~ :' ~
-
~ 1746S8
X-5155 -41-
reacted in dimethylformamide with a molar excess ofl-(lH-tetrazol-5-ylmethyl)-lH-tetrazol-5-thiol at room
temperature to provide p-nitrobenzyl 7-(2-thienyl-
acetamido)-7-methoxy-3-[1-(lH-tetrazol-5-ylmethyl)-lH-
tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylate.
Deesterification of the p-nitrobenzyl ester group
provides the compound of the formula 1 wherein Rl is
hydrogen. The deesterification of the p-nitrobenzyl
ester group is carried out by known reductive cleavage
procedures, for example, by catalytic hydrogenolysis
aver 5~ palladium on carbon in an inert solvent such
as tetrahydrofuran or acetonitrile, or by electrolytic
reductive cleavage at a mercury pool cathode.
Other readily removed carboxylic acid protect-
ing ester groups can likewise be used in the preparationof the 7-methoxy substituted cephalosporins of the
invention. For example, well known carboxylic acid
protecting groups in the art include the diphenyl-
methyl ester group and the p-methoxybenzyl group, both
of which can be removed by treating the ester with tri-
fluoroacetic acid in the presence of anisole preferablv
at a temperature of about 0C. to about 10C. Other
ester groups such as haloalkyl groups, for example,
the 2,2,2-trichloroethyl group and the 2-iodoethyl
group can also be employed and are removed by reduction
with zinc and an acid.
The bis-tetra~olmethyl thiol compounds used
in the preparation of the compounds of the invention
are prepared by reacting l-cyanomethyl-lH-tetrazol-5-
ylthiol with tetramethylguanidinium azide in dioxane
.
:
-1 174~
X-5155 -42-
at the reflux temperature to provide the l-(lH-tetrazol-
5-ylmethyl)-lH-tetrazol-5-thiol. The correspon~ing
2H-tetrazole substituted tetrazole is obtained by
alkylating lH-tetrazole-5-thiol, wherein the thiol
group is protected with a thiol protectin~ group such
as the benzyl group, with a haloaceonitrile such as
chloroacetonitrile in the presence of a base such as
potassium or sodium hydroxide. The alkylation provides
a mixture of 1- and 2-cyanomethyl-5-benzylthio-lH-
tetrazole. The isomeric mixture of the cyanomethylsubstituted tetrazole is then reacted with tetramethyl-
guanidinium azide or with aluminum triazide at elevated
temperatures to provide a mixture of l-(lH-tetrazol-5-
ylmethyl)-lH-tetrazol-5-thiol and 2-(lH-tetrazol-5-
ylmethyl)-2H-tetrazol-5-thiol wherein the thiol group
is protected, for exampie, with a benzyl group. The
isomeric mixture of the bis-tetrazoles is separated
into the individual isomers via HPLC chromatography on
silica gel.
The Cl-C3 alkyl substituted tetrazoles
(R4=Cl-C3 alkyl) are obtained by alkylating the respec-
tive S-protected isomeric tetrazoles wiih a Cl-C3
alkyl bromide or iodide in the presence of a base. The
alkylation affords an isomeric mixture which can be
separated into the individual isomers by ~PLC chroma-
tography. Following the separation of the individual
isomeric tetrazoles or the alkylation products thereof
as described above, the S-protecting group eg. the
benzyl group, is removed from the thiol by electrolytic
reduction a~ a mercury pool cathode.
' :
` ' ~
'' '' ' . ' ' '
.. :.
.
"
1 ~74~68
X-5155 -43-
Alternatively, the l-[l-(Cl-C3 alkyl)-l~-
tetrazol-5-ylmethyl]-lH-tetrazol-5-thiol is prepared
with eth~l 5-chloro-lH-tetrazol-l-acetate. The 5-chloro-
tetrazole acetate is reacted with a Cl-C3 alkylamine to
provide the corresponding N-(Cl-C3 alkyl)amide. The
amide is then reacted with an excess of phosgene at a
temperature of about -5 to about 10C. in an inert
solvent, for example, a halogenated hydrocarbon solvent,
such as metAylene chloride or dichloroethane, to provide
the corresponding N-chlorocarbonyl-N-(Cl-C3 alkyl)amide.
The N-chlorocarbonylamide is then reacted with tet-a-
methylguanidinium azide in dioxane at the reflux tem-
perature to provide the l-[l-(Cl-C3 alkyl)-lH-tetrazol-
5-ylmethyl]-5-chloro-lH-tetrazole. The latter is then
reacted with sodium hydrosulfide to replace the 5-chloro
group with the thiol group.
A preferred group of compounds of tAis invention
are represented by the following general formula.
R5-~-N S\ ~ ~I--N
~OR~O ~ ~ ~ CH2-~
OOH
wherein R is a group represented by the formula
' .'
,
'
,
.. . .
:
: '
1 174~8~
X-5155 ~44~
a~C~
~ y-~
~ - , or ~ ~-
wherein a, a', a'', a''', b, R4 and R6 have the same
meanings as defined above.
Compounds of the above formula which are
especially preferred are represented by tne formula
when R6 is Cl-C3 alkyl and R4 is hydrogen. Especially
preferred compounds are those represented by the formula
when RS is 2-ruryl or the 2-amino-1,3-thiazol-4-yl
group or the tautomeric 2-imino form thereof as shown
by the above structural formula. As is known in the
art, the oximino function in the side chain can h2ve
either the syn or anti form and the compounds in the
syn form are preferred owing to their enhanced activity
over the compounds in the anti form. Examples of the
above compounds include the following.
syn 7~-[a-hydroxyimino-a-(2-furyl)acetamido]-3-[1-
(lH-tetrazol-5-ylmetnyl)-lH-tetrazol-5-ylthiomethyl]-
3-cephem-4-carboxylic acid,
syn 7~-[a-hydroxyimino-a-(2-amino-1,3-thiazol-4-yl)-
acetamido]-3-[1 (lH-tetrazol-5-ylmethyl)-lH-tetrazol-
5-yl~hiomethyl]-3-cephem-4-carboxylic acid,
, :
1 174B68
X-5155 _45_
~y~ 7~-[a-methoximino-a-(2-amino-1,3-thiazol-4-yl)-
acetamido]-3-[1-(lH-tetrazol-5-ylmethyl)-lH-tetrazol-
5-ylthlomethyl]-3-cephem-4-carboxylic acid,
syn 7~-[a-methoximino-a-(2-amino-1,3-thia701-4-yl)-
acetamido]-3-[1-methyl-lH-tetrazol-5-ylmethyl)-lH-
tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylic acid,
syn 7~-[a-hydroxyimino-a-(2-amino-1,3-thiazol-4-yl)-
acetamido]-3-[1-(lH-tetrazol-5-ylmethyl)-lH-tetrazol-
5-ylthiomethyl]-3-cephem-4-carboxylic acid,
syn 7~-[a-methoximino-a-(2-amino-1,3-thiazol-4-yl)-
acetamido]-3-[2-(lH-tetrazol-5-ylmethyl)-2H-tetrazol-
5-ylthiomethyl]-3-cephem-4-car~oxylic acid,
and the pharmaceutically acceptable non-toxic salts
thereof.
Illustrative of other compounds of the inven-
tion represented by formula I are the ~ollowing:
7~-benzamido-3-[1-(lH-tetrazol-5-ylmethyl)-lH-tetrazol-
5-ylthiomethyl]-3-cephem-4-carboxylic Acid,
7~-phenylacetamido-3[1-(lH-tetrazol-5-ylmethyl)-lH-
tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylic acid,
7~-phenoxyacetamido-3-[1-(1-methyl-lH-tetrazol-5-
ylmethyl)-lH-tetrazol-5-ylthiomethyl]-3-cephem-4-
carboxylic acid,
7~-phenoxyacetamido-7a-methoxy-3-[l-(lH-tetra701-5-
~: yl)-lH-tetrazol-5-ylthiomethyl]-3-caphem-4-carboxylic
acid,
7~-phenoxyacetamido-3-12-(lH-tetrazol-S-ylmethyl)-2H-
tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylic acid,
:`
i
.
.. ~ .
:
~ ' ' :
:
~ 174~68
X-5155 -46-
7~-(2-thienylacetamido)-3-[1-(lH-tetrazol-5-ylmethyl)-
lH-tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylic acid,
7~-(2-thienylacetamido)-7a-methoxy-3-[l-(1-ethyl-lH-
tetrazol-5-ylmethyl)-lH-tetrazol-5-ylthiomethyl]-3-
cephem-4-carboxylic acid,
7~-phenylmercaptoacetamido-3-[1-(lH-tetrazol-5-
ylmethyl)-lH-tetrazol-5-ylthiomethyl]-3-cephem-4-
carboxylic acid,
7~-(D-phenylglycylamido)-3-~1-(1-methyl-lH-tetrazol-
5-ylmethyl)-lH-tetrazol-5-ylthiomethyl]-3-cephem-4-
carboxylic acid,
7~-(D-mandelamido)-7a-methoxy-3-[1-(lH-tetrazol-5-
15 ylmethyl)-lH-tetrazol-5-ylthiomethyl]-3-cephem-g-
carboxylic acid,
7~-(D-a-carboxyphenylacetamido~-3-[l-(lH-tetrazol-5-
ylmethyl)-lH-tetrazol-5-ylthiomethyl]-3-cephem-4-
carboxylic acid,
7~-(D-a-sulfophenylacetamido)-3-~l-(1-methyl-lH-
tetrazol-5-ylmethyl)-lH-tetrazol-S-ylthiomethyl]-3-
cephem-4-carboxylic acid,
7~-~a-amino-(2-thienyl)acetamido]-3-~2-(lH-tetraæol-5-
ylmethyl)-2H-tetrazol-5-ylthiomethyl]-3-cephem-4-
carboxylic acid,
7~-[a-(2,3-dihydroxy~enzamido)phenylacetamido]-3-
[l-(lH-tetrazol-5-ylmethyl)-lH-tetrazol-5-ylthiomethyl~-
3-cephem-4-carboxylic acid,
.
,
1 174~68
X-5155 _47_
7~-[a-(2-hydroxypyridin-1-ylcarbonylamino)phenyl-
acetamido]-3-[1-(lH-tetrazol-5-ylmethyl)-lH-tetrazol-
5-ylthiomethyl]-3-cephem-4-carboxylic acid, and
the a-substituted-aminophenylacetamido compounds
represented by the formula
~ LCH2-S
7 O0H
wherein R7 is a group of the formulae
~ ~/CI~ ICHs ~ _ ~H3 /CI ~H3
~ C-N- 7~ CH=CH-~-N~ -CH=CH-~-N-
CH~-NH-~ ~- H ~ CH3-SO2 ~ Or
CH3CH2-N~ ~N
and the pharmaceutically acceptable salts thereof.
The bis-tetrazol-thiomethyl substituted
cephalosporins of this invention are broad spectrum
antibiotics which inhibit the growth of both gram
: 30 positive and gram negative microorganisms which are
~",
`: :
.` , ~
.
~ 174668
X-5155 -48-
pathogenic to man and animals. The antibacterial
activity o~ these compounds was demonstrated in ln
vitro tests carried out by the agar dilution method.
The following Table 2 lists the minimum inhibitory
concentrations of representative compounds of the
invention.
.. . . . . .
.~ :
, ~
,:
. .
~ ~74868
X-5155 -49-
_~ ~ ~~ U~ o o ~ ~ o o
~ ,1
~3
o
~:: ,~ . o ~ ,~ ,
O A A ~ ~
t~ D O O (~I r-l~I ~1 1 1
a~
Q,~
~o 8 c~ ~ .D ~ ~ co ~ u~ ~nco er ~n ~ ,
C~ ~1 ~ ~ ~ N
~1 . ,1 0 0
S~ ~ .
n ~n o
OZ ~V
H ~l ~rl ~u~ oo cl:) ~ ~ I I
~1 ~; ~ O _I a) co ~co ~ ~ ~ ~ I
E~ O ~i ~ _I ~
H P~ _1
H O
E~ ~
~ ~ ~ m
m ~ ~ ~ ~ x u. ~r ~r c~, , u. u,
o ~ _, ~ . ~9 ~I ~ ~ ~
20 E~ m ~ ~ . .
EH~ o
~ ~ ~ ~ (~
~ .,1 ~tO ~~1 ~~ ~'~D O S: ~1 ~D
0 1:1 1~ _IO ~H H O ~~D ~D ~ Ul ~ I`
x ~ ~c ;n~ ~i u ~ a' ~ ~
'~ S ' Ul
~: " ~n
H .q ~1 al
o~
e ~ N
oa~ ~ 1
~ P.
u~
e
,~ u Ut~ U t~ u~
~: O O
U O O O ~1
t~ O U t) U rl
~ --I - ~: O 0 0: S: :
h S ,C ~ ~ ~ O
~) n~ ~ 4 Ll
,
~ u~
.
~.
,
. .
,
": . . ,, ,: .
~ 174668
X-5155 _50_
_ V ~ Ir) u~ u~ ~ CO U~ u~ u7 ~ er u~ In
~1 N NN N O N N D N
_ ~ N N O~ O N ~`J N ~ N
O . . ..
ta
~J N ON O~1 0N N ~I N N
a~
~ a u~ N Ir~ N In ~I C~
~ N N ~I N N N
~ O
E~ ~i
,ÇI ~ _I Ntn cc~ N~ Ir~ ~ N 0 CO _I N
.,1 N ~J N N N
.C , ,~
~ H ~\ A
C: ~i ~ ~ ~N a~In X1~1 N ~O 00 CO ~1 _I
~i N ~ r~ N N
.,1 A A A
~¢ X co et~ o oo --~ N
N N N N
,¢ ,~
E~
.,.~ cr O ~ 5~ ~D ~ CO N 1~ U~ .¢ ~r In
f~ Z ~ ~,N ~¢ ~D ~ ~I m u~
z c~ ; x o m ~
. ~ F~ J N X ~_1
, :
0: : ~:
,1 s~ O
,~
e ~ u
.,, o ~ a
U~
s
~ 0
O ~1 ~ 0
.~ o ~ o
.i ,~ s~
~r~ S U~
~ U~
,
~ 174668
X-5155 -51-
_ ~ ~ 1
e
U
~ E~l ~ ~ ~u. ~ u. ~ ~ ~ ~ ,,0
~ ~ ~ ~ . ~ . o ~ ,~
c ,~
o
~ ~ ~ ~ O ~ ~ ~ r~ ~
~ ~ ~ a~ ~r 0
ul o
G~ ~
,~ ~ ~ ~ ~ ~ . ~ _,
_~ A A A A A
~ m
O~ ~ ~ ~ ~ ~ ~ ~D
t) ~ ,/ ~
~ A A A /~ A A
2 0 ~ A A A A A
,1 ~ a~ x ~ ~ ~ ~ r~
~ ~ ~ --l ~ w ~
u~ ~ ~n x
~ x x ~
:: o
a~ O
~ ~ ~ ~ u
o 8 ~
. & ~3 ~ o
u~ m
,
~,
~ 1~4668
X-5155 -52-
/A. 7~-(2-Thienylacetamido)-7a-methoxy-3-[1-(lH-
tetrazol-5-ylmethyl)-lH-tetrazol-5-ylthiomethyl]-
3-cephem-4-carboxylic acid
B. 7~-(2-Thienylacetamido)-3-[1-(1-methyl-lH-
tetrazol-5-ylmethyl)-lH-tetrazol-5-ylthiomethyl]-
3-cephem-4-carboxylic acid
C. 7~-(D o-Formylmandelamido)-3-[1-(lH-tetrazol-5-
ylmethyl)-lH-tetrazol-5-ylthiomethyl]-3-cephem-
4-carboxylic acid
D. 7~-[D ~-(4-Ethylpiperazin-2,3-dione-1-ylcarbonyl-
amino)-~-(4-hydroxyphenyl)acetamido]-3-[iH-tetra~ol-
5-ylmethyl)-lH-tetrazol-5-ylthiomethyl]-3-cephem-
4-car~oxylic acid
E. syn 7~-[~-Methoximino-a-(2-amino-1,3-thiazol-4-yl)-
acetamido]-3-[1-(lH-tetrazol-5-ylmethyl)-lH-
tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylic acid
F. syn 7~-[a-Methoximino-~-(2-amino-1,3-thiazol-4-yl)-
acetamido]-3-[1-(1-methyl-lH-tetrazol-5-ylmethyl)-
lH-tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylic acid
G. syn 7~-[a-Methoximino-a-(2-amino-1,3-thiazol-4-yl)-
acetamido]-3-[2-(lEI-tetrazol-5-ylmethyl)-2H-tetrazol-
5-ylthiomethyl]-3-cephem-4-carboxylic acid.
/H. influenzae sensitive strain
3/H. influenzae resistant strain
The data in TABLE 2 were obtained in the agar
dilution test method using Mueller-Hinton agar containing
two percent Supplement C ~Dirco). Serial two-fold
dilutions were run with the highest concentration on
test at 128 mcg/ml.
Compound E (TAB~E 2), a preferred compound of
the invention, was administered tG mice infected with
various pathogens to determine the compound's effective
dose (ED50). Listed below in TABLE 3 are the E~50 values
determined with the indicated infectious organism.
'
1 174568
X-5155 -53_
TABLE 3
In Vivo ACTIVITY OF COMPOUND E IN MICE
ED (mg/kg x 2) s.c.
Bacterla (straln) 50
Escherichia coli (EC14) 0.16
Proteus vulgaris (PV2S) 0.12
Serratia marcescens (SE3) 1.0
Staphylococcus aureus (3055) 7.~
Streptococcus pyogenes (C203) 0.21
/Subcutaneous doses at 1 and 5 hours post infection
Compound E was also effective in the treat-
ment of ampicillin-resistant Haemophilus influenzae
meningitis in infant rats. Compound E was found to be
present in effective levels in the cerebro-spinal fluid
of rats administered 20 mg/kg. s.c. of the compound.
The compounds of this invention are ~ffective
in the treatment of infectious diseases in man and
animals when administered by the parenteral route at an
effective dose of between about 50 mg/kg and about
500 mg/kg. The compounds can be administered intra-
muscularly or intravenously in suitable formulations.
For intramuscular preparations, a sterile formulation
of a suitable salt form of the compound of the inven-
tion can be formulated as, for example, the sodium salt
in a pharmaceutical diluent such as Water for Injection,
physiological saline, or as a suspension in a suitable
pharmaceutically acceptable oil ~ase such as an ester
of a long chain fatty acid such as ethyl oleate. For
intravenous admini~tration, a pharmaceutically accepta-
.
.~ . .:
~ ' : ` ' '
~ 174668
X-5155 -54_
ble salt form of a compound of the invention is formu-
lated in a physiological fluid such as Ringer's solution,
5% dextrose, or other suitable physiological fluid. As
is common in antibiotic therapy, the amount of antibi-
otic administered is in general dependent upon theseverity of the infection, the sensitivity of the
particular patient, and the type of microorganism or
microorganisms involved in the infection. Usually the
compounds are administered from 2 to 4 times daily
until the infection is controlled. Accordingly, this
invention also relates to a pharmaceutical formulation
comprising a compound of the invention as represented
by the formula 1 wherein Rl is hydrogen or a pharmaceu-
tically acceptable salt and a physiological carrier.
The following examples further illustrate
the present invention. In the examples, HPLC means
high performance liquid chromatography and, unless
indicated otherwise, a Waters and Associates Model 500
(silica gel) column was employed. The nuclear magnetic
resonance spectra (NMR) were obtained on a Varian
Associates Model T-60 Spectrometer; a ~eol Model
FX-9OQ was used for 90 MHz spectra; and a Jeol Model
PFT-100 Spectrometer was used for 100 MHz spectra.
Trimethylsilane (TMS) was used as the standard in the
Z5 NMR spectra. The abbreviations used in the xecitation
of the NMR spectra ha~e the following meanings: s =
singlet; q = quartet; m = multiplet; d = doublet; t =
triplet.
In the examples, abbreviations are used for
certain solvents as follows: THF = tetrahydrofuran;
.
.
.
1 174~68
X-5155 _55_
DMF = dimethylformamide; DMAC = dimethylacetamide;
MSA = trimethylsilylacetamide; BSA = bis-trimethyl-
silylacetamide.
Example 1
7-Amino-3-[1-(1-methyl-lH-tetra~ol-5-ylmethyl)-lH-
tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylic acid.
A solution of 1.54 g. of 7-aminocephalospo-
ranic acid and 1.0 g. (5 mmole) of l-(l-methyl-lH-
tetrazol-5-ylmethyl)-lH-tetrazol-5-thiol in 7.0 ml. of
glacial acetic acid was heated in an oil bath to a
temperature of about 65-70C. To the hot reaction
mixture were added 3.6 ml. of boron trifluoride etherate,
and the reaction mixture was heated at a temperature of
65-70C. for one hour with stirring. The reaction
mixture was then cooled to room temperature and 10 ml.
of water w~re added. The reaction mixture was stirred
for 15 minutes after water addition, was filtered, and
then chilled in an ice bath. The pH of the cold Mixture
was adjusted to p~ 4.5 with concentrated ~monium
hydroxide. The precipitate which formed was filtered,
washed repeatedly with water, with acetone, and finally
with dietnyl ether. The off-white solid product was
vacuum dried for one hour at 40C. There was obtained
1.5 g. (80~ yield) of the title compGund.
The following analytical data were obtained
for the product.
Elemental analysis calculated for C12~14N10O3S2:
Theory: C, 35.12; ~, 3.44; N, 34.13; S, 15.62
Found: C, 35.62; H, 3.53; N, 31.63; S, 14.52.
.
' ~, .
~ .:
: ' ~
- :
. ~ :
,.
~ 174~68
X-5155 -56-
IR (KBr) carbonyl absorption at 1800 cm 1.
NMR (T-60, NaHC03/D20~
Example 2
7~-[a-Methoximino-a-(2-amino-1,3-thiazol-4-yl)aceta-
mido]-3-[1-(1-methyl~ thiazol-5-ylmethyl)-lH-tetrazol-
5-ylthiomethyl]-3-cephem-4-carboxylic acid.
To a solution of 5.9 g. (15.6 mmole) of
7-am~no-3-[1-(1-methyl-lH-tetrazol-5-ylmethyl)-lH
tetraæol-5-ylthiomethyl]-3-cephem-4-carboxylic acid in
60 ml. of acetone and 60 ml. of water were added 6.2 g.
(17.0 mmole) of a-methoximino-a-(2-amino-1,3-triazol-5-
yl) acetic acid hydroxybenzotriazole ester, and the pH
of the solution was maintained at about 7.5 with a 45%
~5 potassium phosphate solution. The reaction mixture was
~tirred at room temperature for about 12 hours after
which the reaction mixture was evaporated ln vacuo to
remove the acetone solvent. The aqueous residue (pH
7.0) was extracted twice with 100 ml. portions of ethyl
acetate and was then filtered. The aqueous layer was
chilled to a temperature of about 0C., and the pH
adjusted to 2.5 with 20% hydrochloric acid. The acyla~
tion product which formed as a precipita~e was filtered
and washed repeatedly with water. The product was
vacuum dried at room temperature.
The product obtained above was purified by
high performance liquid chromatography as follows. A
solution of 7.0 g. of the compound in 200 ml. of 0.05 M
ammonium acetate containing 0.3 ml. of ammonium hy-
droxide was placed on a Waters and Associates Model 500
,
'
.
'
1 ~74~68
X-;155 -57_
HPLC and the chromatogram was run on C18 reverse phase
silica gel initially with a mixture of 12% acetonitrile:-
2% acetic acid:86~ water, and then with the mixture 15
acetonitrile:2~ acetic acid:83% water. Thirty-two,
250 ml. fractions were collected. The chromatogram was
monitored via analytical ~PLC. Fractions 15-22 were
combined and concentrated ln vacuo. The product precipi-
tated from the concentrate and was filtered and washed
with water. On drying, 1.4 g. of the product were
obtained.
The following nuclear magnetic resonance
spectrum of the purified product was obtained.
NMR (100 MHz, DMSO-d6) o 3.6~ (m, 2H,
C-2 methylene), 3.85 (s, 3H, oxime methyl), 4.15 (s,
3H, N-methyl [tetrazol]), 4.35 (m, 2H, C-3' methylene),
5.13 (d, 1~, C-6 H), 5.76 (q, lH, C-7 H), 6.1 ~s, 2H,
methylene), 6.74 (s, lH, thiazol H), 7.20 (broad s,
2H, thiazole amino H), and 9.58 (d, lH, amide H) ppm.
Example 3
7-Amino-3-11-(lH-tetrazol-5-ylmethyl)-lH-tetrazol-5-
ylthiomethyl]-3-cephem-4-carboxylic acid.
A suspension of 5.3 g. (29 mmole) of l-(lH-
tetrazol-5-ylmethyl)-lH-tetrazol-5-thiol and 6.8 g.
(25 mmole) of 7- aminocephalosporanic acid were suspended
in 38 ml. of glacial acetic acid, and the suspension
was heated at a temperature of about 62 to about 65C.
in an oil bath. To the hot suspension were added
16.6 ml. of boron trifluoride etherats (distilled) and
a nearly complete solution was obtained. The mixture
1 174668
X-5155 -58-
was heated for 1 hour at a temperature of about 60 to
about 65C. with stirring. The reaction mixture was
cooled and stirred for 1 hour at room temperature and
45 ml. of water were added. The diluted reaction
mixture was then cooled to 0C. ~nd the pH adjusted to
pH 4.0 with concentrated ammonium hydroxide. The
product precipitated and was washed with water, with
acetone, diethyl ether, and was dried in a vacuum oven
for 2 hours at a temperature of 50C. There were
obtained 8.0 g. of the product (88~ yield) as a light
beige powder.
NMR (T-60, sodium bicarbonate/D2O) ppm.: 4.3
(m, 2H, C-3' methylene) and 5.9 (s, 2H, methylene bridg-
ing tetrazole rings) delta.
Example 4
7~-~a-Methoximino-a-(2-amino-1,3-thiazol-4-yl)acetamido]-
3-[1-(lH-tetrazol-5-ylmethyl)-lH-tetrazol-5-ylthiomethyl]-
3-cephem-4-carboxylic acid.
To a solution of 7.5 g. (20.6 mmole) of the
bis-tetrazolmethyl nucleus prepared as described in the
preceding example in 75 ml. of water and 75 ml. of
acetone were added 8.6 g. (23.5 mmole) of a-methoximino-
a-(2-amino-1,3-thiazol-4-yl)acetic acid hydroxybenzo-
txiazole ester. The acylation mixture was maintained
at a pH of 7.5 with a 45% solution of potasslum phos-
phate. The reaction mixture was stirred at room tempera-
ture for about 12 hours and evaporated ln vacuo to
remove the acetone solvent. ~rhe aqueous residue was
filtered and cooled in an ice bath. The pH of the
~ ,~
3. 17466~
X-5155 -59_
aqueous phase was adjusted to pH 2.4 wi~h 20~ hydro-
chloric acid. The product precipitated as a thick
yellow precipitate, was filtered, washed repeatedly
with water, and was air-dried. There were obtained
6.5 g. of the product as a light beige solid.
The product was purified hy HPLC chromatog-
raphy as follows. Approximately 12 g. of the crude
product prepared as described above were dissolved in
lO0 ml. of water by adding a 45~ aqueous solution of
potassium phosphate until the pH reached 5.4. This
solution was pumped through a Waters and Associates
Model 500 HPLC col~lmn (silica gel) over a reversed
phase cartridge using the following systems.
A. 8~ Acetoni-rile:2~ acetic acid:90~ water
(4 liters)
B. 10% ~cetonitrile:2% acetic acid:88% water
(6 liters)
Forty, 250 ml. fractions were collected and the fractions
were monitored by ultraviolet absorption. Fractions
11-22 were combined and lyophilized to 2.5 g of an
amorphous white solid.
NMR (360 MHZt DMSOd6): ~ (ppm). 3.65 (m, 2H,
C-2 methylene), 3.85 (5~ 3H, oxime methyl, syn), 4.35
(m, 2H, C-3' melhylene), 5.13 td, lH, C-6H), 5.80 (q,
lH, C-7H), 6.02 (m, 2H, methylene between tetrazole
rings), 6.75 (s, lH, thiazoie ring H), 7.24 (broad s,
2H, thiazole amino), and 9.60 (d, lH, 7-amid~ H).
- .
,:
1 174~6~
X-5155 -60-
Example 5
7~-~a-Methoximino-a-(2-amino-1,3-thiazol-4-yl)acetamido]-
3-[2-(lH-tetrazol-5-ylmethyl)-2H-tetrazol-5-ylthio-
methyl]-3-cephem-4-carboxylic acid.
To a solution of 202 mg. (1.1 mmole) of
2-(lH-tetrazol-5-ylmethyl)-2H-tetrazol-5-thiol in 6 ml.
of pH 7 buffer lsolution was obtained with 176 mg.
2.1 mmole, of sodium bicarbonate) were added 460 mg.
(1 mmole) of 7~-[a-methoximino-a-(2-amino-1,3-thiazol-
4-yl)acetamido]cephalosporanic acid, and the solution
was heated in an oil bath for 22 hours at a temperature
of about 55C. to about 60C. After this time, a thin
layer chromatogram of the reaction mixture indicated
that the reaction was about one-half completed. An
additional 100 mg. of the thiol and an equivalent
amount of sodium bicarbonate were added to the reaction
mixture. The reaction mixture was then heated for
about 12 hours at a temperature of 55-60C. The reac-
tion mixture was cooled to room temperature and waswashed twice with 50 ml. portions of ethyL acetate.
The aqueous phase was filtered, and the filtrate cooled
in an ice bath to a temperatur~ of about 0C. The pH
of the aqueous phase was adjusted to 3.2 with vigorous
stirring, and the product precipitated as a yellow-
orange precipitate. The product was filtered, washed
with water, and dried at room temperature in vacuo.
There were obtained 153 mg. of a mixture of the syn
and anti forms of the product. The isomers were
separated over reverse phase silica gel using 15%
~ acetonitrile, 2% acetic acid, 83% water, v:v:v~
,.
.
1 174~
X-5155 -61-
syn Isomer NMR (360 MHz, DMSOd6): ~ 3.65 (m, 2H,
C-2 methylene), 3.85 (s, 3H, oxime methyl), 4.27 (m,
2H, C-3' methylene), 5.13 (d, lH, C-6H), 5.82 ~q,
lH, C-7H), 6.42 (s, 2H, methylene bridging tetrazole
rings), 6.78 (s, lH, thiazole H), 7.29 (broad s, 2H,
thiazole amino;, and 9.61 (d, lH, 7-amide H) ppm.
anti Isomer ~MR (360 MHz, DMSOd6): ~ 3.60 (m, 2H,
C-2 methylene), 3.95 (s, 3H, oxime methyl), 4.27 (m,
2H, C-3' methylene), 4.97 (d, lH, C-6H), 5.76 (q, lH,
C-7H), 6.33 (s, 2H, methylene bridging tetrazole rings),
7.14 (broad s, 2H, thiazole amino H), 7.46 (s, lH,
';hiazole H), and 9.43 (d, lH, 7-amide H) ppm.
Example 6
7-(2-Thienylacetamido)-3-[1-(lH-tetrazole-
5-ylmethyl)-lH-tetrazole-5-ylthiomethyl]-3-cephem-4-
carboxylic acid.
~ solution of 808 mg. (2 mmole) of sodium
7-(2-thienylacetamido)-3-acetoxymethyl-3-cephem-4-
carboxylate (sodium cephalothin), 368 mg. (2 mmole)
of l-(lH-tetrazole-5-ylmethyl)-lH-tetrazole-5-thiol,
and 168 mg. (~ ~mole) of sodium bicarbonate in 16 ml.
of water was hea~ed in an oil bath for 7 hours at a
temperzture between about 55C. and about 60C. The
reaction mixture was concentrated under vacuum to a
volume of about 8 ml. and injected onto reverse phase
silica gel preparative HPLC. Separation was achieved by
first using 15~ acetonitrile:2~ acetic acid by volume
in water. Multiple 20 ml. fractions were collected and
after about 90 fractions were collected, the elueht was
:
.
1 17~68
X-~155 -62-
changed to 20% acetonitrile in water. Fractions 133-153
containing the product were combined and evaporated.
There were obtained 350 mg. of crystalline product as
the p-nitrobenzyl ester. Fractions 181-193 contained
the starting material sodium cephalcthin.
The following data were obtained for the
crystalline product.
NMR (100 MHz, DMSO-d6): ~ 3.63 (m, 2~, C-2
methylene), 3.73 (s, 2H, thiopheneacetyl methylene),
4.32 (m, 2H, C-3' methylene), 5.04 (d, lH, C-6H),
5.63 (q, lH, C-7H), 5.97 (s, 2H, tetrazole methylene),
6.8-7.4 (m, 3H, thiophene H), and 9.08 (d, lH, 7-amide
H) ppm.
Elemental analysis:
Theory: C, 39.22; H, 3.10; N, 26.91
Found: C, 38.86; H, 3.33; N, 25.11.
IR (KBr) ~-lactam carbonyl absorption at 1775 cm 1.
Field Desorption Mass Spectrum: MW - 521.
Exampie 7
7-(2-Thienylacetamido)-7-methoY.y-3-[1-(lH-tetrazole-5-
ylmethyl)-lH-tetrazole-5-ylthiomethyl]-3-cephem-4-
carboxylic acid.
A solution of 3.1 g. (5.3 mmole) of p-nitro-
~5 benzyl 7-(2-thienylacetamido)-7-methoxy-3-bromomethyl-
3-cephem-4-carboxylate and 1.38 g~ (7.5 mmole) of
l-(lH-tetrazole-5-ylmethyl)-lE-tetrazole-5-thiol in
20 ml. of DMF was stirred at room temperature for about
16 hours. The reaction mixture was poured into
150 ml. of a mixture of equal volumes of lN hydrochloric
1 174~68
X-5155 -63-
acid and ethyl acetate. The organic layer was separated,
washed twice with lN hydrochloric acid, once with brine,
and was dried over sodium sulfate, filtered, and
evaporated to dryness. There were obtained 3 g. of
the impure product as a light brown foam. The product
was chromatographed over 75 g. of silica gel using
1 liter of methylene chloride followed by two liters
of methylene chloride containing 2% methyl alcohol for
elution. The column was maintained under nitrogen
pressure during elution. The fractions containing
the product were combined and evaporated to dryness.
The residual oil was dissolved in ethyl acetate and
the solution was filtered. The filtrate was concentrated
under vacuum to dryness. There was obtained 0.84 g.
of the product.
The above product was deesterified by
electrolytic reduction as follows: A solution of 700 my.
(1 mmole) of the p-nitrobenzyl ester in 30 ml. of lN
sulfuric acid in DMF was placed in an electrolysis cell
comprising a mercury pool cathode ar.d a platinum wire
anode. The electrodes were separated by a sintered
glass frit. The reduction was carried out at a poten-
tial of -C.64 v over 3 hours. The reduction solution
was remo~-ed from the cell and was poured into 100 ml.
of a mixture of equal volumes of ethyl acetate and lN
hydrochloric acid. The organic layer was separated and
washed twi~e with lN hydrochloric acid. The product
was then extracted from the organic layer with 50 ml.
of aqueous sodium hydroxide, pH 7.5 and the aqueous
exLract was washed with ethyl acetate. The aqueous
' ' '
'' ' '; ' ' ~,
:
', '
.
11748~
x-5155 -64-
phase was layered with fresh ethyl acetate and acidlfied
to pH 2.2 with hydrochloric acid. The organic layer
was separated, washed twice with lN hydrochloric acid,
with brine, dried over sodium sulfate and evaporated
to dryness. There were obtained 290 mg. of the
product in the free acid form as a yellow foam.
The product w~s purified as follows: The
produst, 270 mg. was dissolved in O.OSM ammonium
acetate by adding two drops of concentrated a~monium
hydroxide to the suspension o, the product and the
solution was filtered and injected onto a reverse phase
silica gel HPLC preparative column. The column was
initially eluted with 12% acetonitrile:2% acetic
acid:water and then with 15% acetonitrile:2~ acetic
acid:water. ~ulti.ple 20 ml. fractions were col].ected
and fractions 72-89 were combined and lyophilized.
There were obtained 75 mg. of the product as a dry
powder.
The following data were obtained for the
product.
IR (KBr) ~-lactam carbonyl absorption at 1775 cm 1
Field Desorption Mass Spectrum: MW = 551.
NMR (100 MHz, DMSO-d6): ~ 3.34 (s, 3H, 7-methoxy"
3.51 (m, 2H, C-2 methylene), 3.80 (s, 2H, thiophene-
acetyl methyle~.e), 4.29 ~m, 2H, C-3' methylene), 5.05
(s, lH, C-6H), 5.97 (s, 2H, tetrazole methylene),
6.9-7.4 (m, 3H, thiophene ring H~, and 9.42 (s, lH,
7-amide H) ppm.
,: :
.
.
.
~l74ssa
X~5155 -65-
Example 8
7-(O-Formylmandelamido)-3-[1-(lH-tetrazole-5-ylmethyl)-
lH-tetrazole-5-thiol]-3-cephem-4-carboxylic acid.
To a solution of 1.01 g. (5.5 mmole) of 1-
(lH-tetrazole-5-ylmethyl)-lH-tetrazole-5-thiol in 15 ml.
of pH 7.0 buffer containing 0.88 g. of sodium bicarbonate
were added 2.28 g. (5 mmole) of sodium 7-(O-formylmandel-
amido)-3-acetoxymethyl-3-cephem-4-carboxylate and the
solution was heated with stirring at a temperature of
about 60C. to about 65C. for 16 hours. The reaction
mixture was cooled to room temperature and was concen-
trated to a volume of 6.5 ml. by evaporation in vacuo.
The concentrate was chromatographed in silica gel
reverse phase HPLC using 12% acetonitrile:2~ acetic
acid:water as eluent. Multiple 20 ml. fractions were
collected and monitored by W for the cephem chromophore
content. Fractions 94-107 were combined and lyophilized.
There were obtained 120 ml. of the title compound.
NMR (90 MHz, DMSO-d6): signals at ~ 3.84 (m, C-2
methylene), 4.54 (m, C-3' methylene), 5.26 (d, C-6H),
5.32 (s, methine H of mandelamido side chain), 5.94 (q,
C-7H), 6.24 (s, tetrazole methylene), 7.4-7.8 (m,
phenyl H), and 9.04 (d, 7-amine H) ppm.
Example 9
7-[a-~4-Ethylpiperazin-2,3-dione-1-ylcarbonylamino)-a-
(4-hydroxyphenyl)acetamido]-3-~1-(lH-tetrazole-5-
ylmethyl)-lH-tetrazole-S-ylthiomethyl]-3-cephem-4-
carboxylic acid.
.
. - . . :
,
~. : s
:' , . . . .
1 17466~
X-S155 -66-
To a suspension of ~.3 g. (50 mmole) of 4-
hydroxyphenylglycine in 75 ml. of tetrahydrofuran were
added 25 ml. of bis-trimethylsilylacetamide and the
suspension was stirred at room temperature for 15 min-
utes and then at 75C. for 2.5 hours to form a solution.
The solution was cooled to 0C. and 25 ml. of propylene
oxide were added. Next a solution of 11 g. of 4-ethyl-
piperazin-2,3-dione-l-ylcarbonyl chloride in 100 ml. of
THF was added and the reaction mixture was stirred for
1 hour at 0C. and at room temperature for 4 hours.
Methyl alcohol, 40 ml., were added to the reaction
mixtur~ and after stirring for 15 minutes the mixture
was evaporated to dryness. The gummy residue was
dissolved in a mixture of ethyl acetate-aqueous sodium
bicarbonate and the aqueous layer was separated, washed
with ethyl acetate, filtered, and cooled to 0C. The
cold solution was layered with 200 ml. of fresh ethyl
acetate and the pH adjusted to pH 1.9 with concentrated
hydrochloric acid. The organic phase was separated,
combined with an ethyl acetate wash of the acidified
aqueous phase and dried over sodium sulfate. Evapora-
tion of the dried organic phase gave 6.7 g. (40% yield)
of a-(4-ethylpiperazin-2,3-dione-l-ylcarbonylamino)-4-
hydroxyphenylacetic acid.
2S A solution of 670 mg. (2 mmole) of the acid
and 306 mg. of hydroxybenzotriazole in 10 ml. of THF
and containing 0.5 g. molecular sieve was stirred at
room temperature for 1 hour, the solution cooled to
0C. and 450 mg. of dicyclohexylcarbodiimide wer~ added
with continual stirring for 20 minutes. ~he reaction
.
1 174668
X-5155 -67-
mixture was filtered to remove the sieve and dicyclo-
hexylurea, and the filtrate was added to a 0C. solution
of 750 mg. (2 mmole) of -7-amuno-3-~1-(LH-tetrazol-5-y ~ thyl)-IH-tetrazol-5-
ylthio methyl~-3-cephem-4-car~lic acid in 5 ml. of THF and 5 ml. of water
containing 0.52 ml. of triethylamine. The reaction
mixture was stirred at room temperature for about 16
hours and was evaporated in vacuo to remove the THF.
The concentrate was diluted w th 20 ml. of water and
the pH adjusted to pH 7.0 with a 45% aqueous solution
of potassium phosphate. The acifidied solution was
washed with 20 ml. of ethyl acetate, filtered, cooled
to 0C. and acidified to pH 1.~ with 20~ hydrochloric
acid. The product precipitated as a gum and the super-
natant was decanted. Fresh water, 25 ml., were added
to the gum and the mixture sonicated for 15 minutes
until the product formed as a light brown solid. ~he
product was filtered, washed with water and dried in
vacuo at room temperature. Thare were obt~ined 750 mg.
of th~ title compound.
The product was puri.ied as follows. A
suspensior of 700 mg. of the above product in 6 ml. of
O.O5M am~onium acetate was treated with ~ arop~ of
concentra~ed ammonium hydroxide to form a solu'ion.
The solution was injected onto a siLicz gel (C-18)
reverse phase HPLC and chromatographed at 2~0 psi using
12:2:B6; acetonitrile:acetic acid:water; v:v:v as
eluent. Multiple fractions of about 20 ml. were col-
lected. Fractiors 104-120 were combined ~nd evaporated
to remove the acetonitrile. The aqueous residue was
lyophilized to provide 87 mg. of the purified product
containing a small amount of acetic acid.
'
1174~68
X-5155 -68-
NMR (90 MHz, DMSO-d6): ~ 1.25 (t, protons of
methyl moiety of the 4-ethyl group), 3.4-4.2 (m, methyl-
ene protons of piperazine and C-2 methylene), 4~46 (m,
C-3' methylene), 5.14 (d, C-6H), 5.62 (d, methine H of
4-hydroxyphenylacetyl group), 5.88 (q, C-7H), 6.08 (s,
tetrazole methylene), 7.10 (m, 4-hydroxyphenyl H), 9.50
(d, a-amide H), and 9.91 (d, C-7 amide H~ ppm.
Example 10
syn 7~-[a-(Methoximino)-a-(2-furyl)acetamido]-3-[1-
(lH-tetrazol-S-ylmethyl)-lH-tetrazol-5-ylthiomethyl]-
3-cephem-4-carboxylic acid.
A solution of sodium 7~-[a-(methoximino)-a-
(2-furyl)aceta~ido]-3-acetox~nethyl 3-cephem-4-
carboxylate, an equimolar amount of 1-(1~-tetrazol-5-
ylmethyl)-l~.-t~razole-S-thiol and an equimolar amount
of sodium bicarbonate in water is heated for about 24
ho~lrs at a temperature of about 55C. The reaction
mixture is concentrated under reduced pressure and the
product is isolated from the concentrate by ~ilica gel
reverse phase dPLC.
Preparation Examples
The following preparations illustrate the
preparation of the bis-tetrazol~ methyl thiols used to
prepare the cephalosporin compounds of the invention.
Preparation Example 1
Preparation of l-cyanomethyl-lH-tetrazol-5-ylthiol
A. Ethyl azidoacetate
To a solution of ~90 g. (4 moles) of ethyl
chloroacetate in 1500 ml. of acetonitrile were added
,.
:. . . .
, , ~ : ~ ' '' '
:
'
''. :
-
117 466~
X-5155 -69-
260 g. (4 moles) of sodium azide, and the mixture washeated at the reflux temperature for 20 hours. After
heating, the reaction mixture was poured into 1 liter
of water with stirring for 1/2 hour. The organic phase
was separated from the aqueous phase and evaporated in
vacuo to dryness. The yellow residual oil was dissolved
in 1200 ml. of diethyl ether and the solution was dried
over magnesium sulfate. Evaporation of the diethyl
ether in vacuo gave 391 g. (76% yield) of ethyl aæido-
acetate.
B. Ethyl 5-chloro-lH-tetrazol-l-ylacetate
A mixture of 130 g. (l mole) of ethyl azido-
acetate prepared as described in part A and 96 g.
(1.56 mole) of cyanogen chloride was heated at a tem-
perature of 125C. for 20 hours. After the reaction
mixture had cooled, the reaction product mixture was
dissolved in ethyl acetate, and the solution was fil-
tered and evaporated ln vacuo yiel~ing a yellow crystal-
line mass of product. The yellow crystals were recrys-
tallized from aqueous ethyl alcohol and gave 14g g.
(78~ yield) of ethyl 5-chloro-lH-tetrazol-l-ylacetate
as pale yellow crystals melting at about 57-60C.
C. Ethyl 5-thiol-lH-tetrazol-l-ylacetate
A solution of 209 g. of the chlorotatrazole
ester, prepared as described in part B above, and
250 g. of sodium hydrosulfide in 5 liters of ethyl
alcohol was heated at the reflux temperature for 24
hours. Arter heating, the reaction mixture was acidi-
fied with concentrated hydrochloric acid, and the
1 174668
X-5155 -70-
volume of the acidified mixture was reduced to 1/4 theoriginal volume by evaporation ln vacuo. The concen-
trate was extracted with ethyl acetate, the extract was
dried and evaporated to dryness under reduced pressure.
The residual product was recrystallized from toluene-
methylene chloride-hexane and gave 129 g. of the product.
D. S-Thiol-lH-tetrazol-l-ylacetamide ammonium
salt
A solution of 20 g. (0.106 mole) of the
tetrazolthiol ester, prepared as described above in
part C, in 320 ml. of concentrated ammonium hydroxide
and 200 ml. of ethyl alcohol containing 500 ml. of
ammonium chloride was heated at the reflux temperature
for about 12 hours. After heating, the reaction mixture
was evaporated ln vacuo, and the yellow crystalline
residue obtained was recr~stallized from hot ethyl
alcohol to yield a first crop of 13.7 g~ ~73~ yield~
of the product as white crystals melting at about 197
to about 199C. after vacuum drying. A second crop of
1.4 g. of the product was obtained which melted at
about 191-193C.
E. l-Cyanomethyl-lH-tetrazol-5-thiol
A suspension of 5.28 g. of the tetrazolamide
ammonium salt, prepared as described above in part ~,
in 90 ml. of methylene chloride ccntaining 14.4 ml.
- of pyridine was cooled to a temperature of about 0C.
To this suspension was added dropwise with stirring
a solution of 4.6 g. (30 mmole) of phosphorus oxychlo-
ride in 40 ml. of methylene chloride. After the addi-
., . ~ .
1 174~&~
X-5155 -71
tion was completed, the reaction mixture was heated at
the reflux temperature for 30 minutes ancl was then
cooled to room temperature with stirring. The reaction
mixture had turned oranye after heating and contained
some precipitate. The reaction mixture was evaporated
to dryness in vacuo and the residue dissolv~d in ethyl
acetate-water, 1:1, v:v. The pH of the solution was
adjusted to pH 2 with 20~ a~ueous hydrochloric acid.
The acidified solution was then extracted twice with
75 ml. portions of ethyl acetate and the extracts
combined. The extract was then washed with 5~ hvdro-
chloric acid, with brine, was dried o~er sodium sulfate
and evaporated ln vacuo. The brown oil was obtained as
a residue and crystallized on standing. Th~ crystals
were vacuum dried at room temperature and yielded after
drying 2.6 g. (61~ yield) of light brown proauct
melting at about 113-114C.
The above reaction ~as ra2eated on a 10.6 g.
batch of the tetrazol amide anr,lonium salt and 3.7 ~. of
the nitrile as off-white crystals melting at a~out
116-118C. were obtainéd.
The following ar.aiytical data ~ere ob~ained
for the srystallille product.
~lemental analvsis calcualted for C~P~3N5$:
Theory: C, 25.53; H, 2.14; N, 49.62.
Found. C, ~5~82; H, 2n40; N~ 49~91~
The mass spectr~ o~ the crystalline product
~ showed a molecular weight of 141 in agreement with the
;, 30 product.
i
--
, .
~17~66~
X-5155 -72-
Preparation E~ample 2
l-(lH-Tetrazol-5-ylmethyl)-lH-tetrazol-5-thiol
A solution of 6.0 g. (42.5 mmole) of l-cyano-
methyl-lH-tetrazol-5-thiol and 10~0 g. (6.3 mmole) of
tetramethylguanidinium azide in 90 ml. of dioxane was
heated at the reflux temperature for 3 hours. After
cooling, the reaction mixture was evaporated to dryness
in vacuo and the residue dissolved in ethyl acetate:water,
1:1. The ethyl acetate layer was separated, and the pH
of the aqueous layer was adjusted to pH 1.8 with 20%
hydrochloric acid. The acidified aqueous layer was
then extracted 3 times with 75 ml. portions of ethyl
acetate, and the extracts were combined. The extract
was then washed with 5% hydrochloric acid, with brine,
was dried over sodium sulfate, and then evaporated ln
vacuo to dryness. The red oil obtained as a residue
crystallized on seeding. The crystals were washed with
ethyl acetate and with diethyl ether and were dried.
There were obtained 3.7 g. of the bis-tetrazolmethyl
thiol melting at about 173C. to about 175~C. The
filtrate from the first crop was evaporated to an oil
and after seeding the oil, 0.3 g. of a second crop
crystalline product was obtained. A third crop o~
0 3 g. was obtained in the same manner. Total yield of
product was 4.3 g. (55% yield).
The NMR spectrum of the product run in DMSO-d~
showed a singlet at 5.9 ppm delta for the protons of
the methylene group bridging the tetrazole rings.
3~
.:' ' ~: ' ' .- ,: -
'`';', :
ll7~6a
X-5155 _73_
Preparation Example 3
l-(l-Methyl-lH-tetrazol-5-ylmethyl)-lH-tetrazol-S-thiol
A. 5-Chloro-N-methyl-lH-tetrazol-l-acetamide
A solution of 19.5 g. ~0.102 mole) of ethyl
5-chloro-lH-tetrazol-l-acetate in 30 ml. of ethyl
alcohol was cooled in a dry ice-propyl alcohol bath,
and methylamine gas was passed into the solution for 5
minutes. The reaction mixture solidified and was
washed with ethyl alcohol and diethyl ether and was
dried on the steam bath. ~here was obtained 13.2 g.
(74~ yield) of the ~-methylamide product as white
crystalline yields melting at about 146 to about 1483C.
The following analytical data were obtained
for the cr~stalline product.
Elemental analysis calculated for C4H6N50Cl:
Theory: C, 27.36; H, 3.44; N, 39.89; Cl, 20.19.
Found: C, 27.59; H, 3.35; N, 39.65; Cl, 20.49.
NMR (DMSO-d6): ~ 2.7 (d, J=5Hz, 3H, amide
methyl), 5.28 s, 2H, CH2),
8.53 (s, broad, lH, N-H) ppm.
Molecular weight via mass spectrum = 175.5
~ a-Methyl-lH-tetrazol-5-ylmethyl)-5-
chloro-lH-tetrazole
To a s~spension of 1.75 g. (10 mmole) of
5-chloro-N-methyl-lH-tetrazol-l-acetamid~, prepared as
described under A above, in 50 ml. of methylene chloride
containing 0.8 g. of pyridine maintained at a temperature
of about 0C. was added with stirring excess phosphene.
After addition was complete, the reaction mixture was
.,
1 174668
X-5155 ~74~
stirred for 10 minutes without further cooling. The
clear solution obtained on reaction was evaporated to
drynes~ at a temperature of about 30C. under reduced
pressure. The residue containing the reaction product
was suspended in 50 ml. of dioxane, and 2.4 g. (15.2
mmole) of tetramethylguanidinium azide were added to
the suspension. The mixture was heated for 2 hours at
the reflux temperature and after stirring overnisht at
room temperature, the reaction mixture was concentrated
to near dryness under reduced pressure. The concentrate
was dissolved in 30 ml. of water forming a pale yellow
solution from which the product crystallized as color-
less needles. The product was filtered and 0.4 g. of
the crystalline product melting at about 138C. to
about 140C. was obtained. A second crop of 0.5 g. of
the product melting at about 136C. to about 139C. was
isolated from the filtrate.
The following analytical data were obtained
for the crystalline product.
Elemental analysis calculated for C4H5N~Cl:
- Theory: C, 23.95; H, 2.51; N, 55.86; Cl, 17.45.
Found: C, 24.17; H, 2.75; N, 55.81; Cl, 17.85
NMR (DMSO d6): ~ 4.27 (s, 3H, N-CH3), 6.33
(s, 2H, CH2) ppm.
; 25 C. l-(l-Methyl-lH-tetrazol-5-ylmethyl~-lH-
tetrazol-5-thiol
To a suspension of 0.5 g. (2.5 mmole~ of the
; 5-chloro-bis-tetrazol, prepared as described in B
above, in 40 ml. of ethyl alcohol was added 0.6 g. of
,
i
.. -
~: ,
1 17466~
X-5155 -75-
sodium hydrosulfide. The mixture was heated at the
reflux temperature for 16 hours, was cooled to room
temperature, and filtered. The filtrate was concen-
trated to near dryness under reduced pressure and
30 ml. of 5~ hydrochloric acid were added. The acidi-
fied concentrate was extracted 3 times with 30 ml. por- -
tions of ethyl acetate, and the extracts were combined
and washed with 5% hydrochloric acid, brine, and dried
over sodium sulfate. The dried extract was concentrated
to a small volume from which the crystalline product
precipitated. The product was recrystallized from
ethyl acetate-hexane, and there was obtained 0.3 g. of
the pro~uct as nearly colorless crystals melting at
about 190C. to about 192C.
The following analytic~l data were obtained
with the crystalline product.
Elemental analysis calculated for C4H6N8S:
~heory: C, 24.24; H, 3.05; N, 56.53.
Found: C, 24.21; H, 3.28; N, 56.43.
NMR (DMSO-d6): ~ 4.22 (s, 3H, CH3), 5.95 (s, 2H,
CH2)~ 10.57 (broad s, lH, SH~.
The molecular weight as determined by mass
spectrum was 198.
Preparation Example 4
2-(lH-Tetrazole-5-ylmethyl)-2H-tetrazole-5-thiol
A. 5-Benzylthio-lH-tetrazole
A solution of 30 g. (0.33 mole) of thio-
semicarbazide and 51 g. (0.40 mole) of benzyl chloride
~ 17466~
X-~155 -76-
in 500 ml. of ethyl alcohol was heated at the reflux
temperature for about 3.5 hours. After heating, the
reaction mixture was evaporated to dryness under reduced
pressure and the residue was dissolved in water. The
solution was washed with ethyl acetate and was added to
a solution of 25 g. (0.36 mole) of sodium nitrite in
50 ml. of water. The solution was stirred for 15
minutPs and then ethyl acetate was added. The organic
layer was separated and washed with water, brine, and
was dried over sodium sulfate. The dried ethyl acetate
solution was evaporated under redused pressure, and the
product obtained as a residue was washed with methylene
chloride and recrystallized from ethyl acetate. There
were obtained 21 g. of the product melting at about
134C. to about 136C.
The following analytical data were obtained
with the prodllct.
Elemental analysis calculated for C8H8N4S:
Theory: C, 49.98; H, 4.19; N, 29.14.
Found: C, 49.81; H, 4.17; N, 28.95.
B. 1- and 2-Cyanomethyl-5-benzylthic-lH-
tetrazol
A solution of 2.7 g. of potassium hydroxide
in 5 ml. of methyl alcohol was added with stirring to a
solution of 7.9 g. (0.041 M) of 5-benzylthio-lH-
tetrazole in 25 ml. of methyl alcohol, and after stir-
, ring the solution for 15 minutes at room temperature,
3.4 g. (0.045 M) of chloroacetonitrile was added. The
reaction mixture was heated at the reflux temperature
for about 12 hours and the white solid which formed was
,
.
~ 1746B~
X-5155 _77_
filtered. The filtrate was concentrated in vacuo to an
oily residue, and the residue dissolved in a mixture of
diethyl ethsr and water. The ether layer was separated
and washed in an aqueous solution of sodium bicarbonate,
water, and with brine, and was dried and evaporated to
dryness under reduced pressure. There were obtalned
3.4 g. of a mixture of 1- and 2-cyanomethyl-5-benzyl-
thio-lH-tetrazol as a reddish oil. The nuclear NMR
spectrum of the oil showed it was a mixture of approxi-
mately 50~ of each of the isomers.
C. 5-Benzylthiol-2-(lH-tetrazol-5-ylmethyl)-
2H-tetrazole
To 70 ml. of dry tetrahydrofuran cooled in an
ice-ethanol bath were added in small portions 4.04 g.
(0.03 mo}e) of anhydrous aluminum chloride~ After
addition was complete 5.85 g. (0.09 mole) of finely
ground sodium azide were added with stirring. After
stirring the mixture for 5 minutes a solution of 3.93 g.
(0.017 mole) of the 1- and 2-cyanomethyl-5-benzylthio-
tetrazole isomeric mixture in 20 ml. of dry tetrahydro-
furan was added and the mixture heated at the reflux
temperature for 24 hours. The reaction mixture was
cooled in an ice-ethanol mixture and acidified by
dropwise addition of 30 ml. of 20% hydrochloric acid.
The acidified mixture was concentrated under reduced
pressure to a volume of about 30 ml. and the concentrate
was extracted with three 30 ml. portions of ethyl
acetate. The extracts were combined, washed with
brine, and dried over anhydrous sodium sulfate. The
dried extract was evaporated under vacuum to dryness
e,. ~
1 ~74~6~
X-5155 -78-
providing 4.5 g. (97% yield) of a mixture of the iso-
meric 5-benzylthio-1-and 2-(lH-tetrazole-5-yimethyl)-
lH- and 2H-tetrazoles as a tan oil. After standing for
several days, crystals formed in the oil. The mixture
was triturated with methylene chloride and filtered to
provide 0.85 g. of cream-colored crystals melting at
about 115C. to about 117C. A second crop of crystals
which weighed 0.2 g. was obtained from the filtrate.
The above preparation was repeated by react-
ing 7.1 g. of the isomeric 5-benzylthio-1- and 2-cyano-
methyl-tetrazole with aluminum azide (formed as described
above with 7.3 g. of aluminum chloride and 10.7 g. of
sodium azide). After heating at the reflux temperature
for 24 hours, the reaction mixture was acidified with
20% hydrochloric acid, evaporated to a volume Qf ahout
60 ml., extracted with ethyl acetate, the extract
washed with brine, dried, and evaporated to dryness.
The residue crystallized on standing. The cream-colored
crystals were suspended in methylene chloride and
filtered to provide 3.5 g. of crystalline material.
The filtrate was evaporated to dryness to prcvide 4.9 g.
of an orange oil.
The nmr spectrum of the crystalline product
run in deuterated DMSO showed mainly one isomer, the
2-isomer, while the ~mr spectrum of the oil showed
mainly the l-isomer.
~ ::
~ 174~68
X-5155 _79_
D. 2-(lH-Tetrazole-5-ylmethyl)-2H-tetra-
zole-5-thiol
5-Benzylthio-2-(lH-tetrazole-5-ylmethyl)-2H-
tetrazole, 175 mg., prepared as described above under
part C, was dissolved in 40 ml. of distilled D~ and
reduced at the mercury pool cathode (14 cm2 Hg pool)
with a platinium wire anode. The electrodes were
separated by a glass frit. The electrolyte was tetra-
ethylammonium perchlorate, 0.lM in the DMF solution of
the substrate. The electrolysis was carried out at -2.7
to -2.85 volts for 500 seconds at -2.80 v. for about
630 seconds.
The reduction product mixture from the one-
electron reduction was evaporated to dryness and the
residue of product dissolved in ethyl acetate. The
solution was washed three times with a 9:1 by volume
mixture of a saturated solution of sodium chloride
and O.lN hydrochloric acid and was dried over anhydrous
magnesium sulfate. The dried solution was concen-
trated ln vacuo and 111 mg. of the title compoundprecipitated from the concentrate. The product was
filtered and dried.
.
